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(19) United States US 201101361.25A1
(12) Patent Application Publication (10) Pub. No.: US 2011/0136125 A1 Parada ValdecantOS et al. (43) Pub. Date: Jun. 9, 2011
(54) METHOD FOR THE IDENTIFICATION AND (30) Foreign Application Priority Data QUANTIFICATION OF MICROORGANISMS USEFULIN BOMINING PROCESSES Aug. 26, 2005 (CL) ................................... 2179-2005
(75) Inventors: Pilar Angelica Parada Valdecantos, Nunoa (CL); Katia Nicole Ehrenfeld Stolzenbach, Las Condes (CL): Igor Alejandro Pacheco Cruz, Recoleta (CL): Alejandro Eduardo Maass Sepúlveda, Penalolen (CL); Andrés Octavio Aravena Duarte, Nunoa (CL); Mauricio Alejandro Gonzales Canales, Penalolen (CL): Servet Martinez Aguilera, La Cisterna (CL)
(73) Assignee: BIOSIGMA S.A, Colina (CL)
(21) Appl. No.: 12/945.407
(22) Filed: Nov. 12, 2010
Related U.S. Application Data (63) Continuation-in-part of application No. 1 1/509,870,
filed on Aug. 25, 2006, now abandoned.
Publication Classification
(51) Int. Cl. CI2O I/68 (2006.01)
(52) U.S. Cl. ....................................................... 435/6.12
(57) ABSTRACT
The present invention discloses a method to identify and quantify environmental microorganisms useful in biomining processes. These microorganisms are basically 10, belonging to Bacteria: Acidiphilium sp., Leptospirillum sp., Sulfobacil lus sp., Acidithiobacillus ferrooxidans and Acidithiobacillus thiooxidans, and Archaea: Acidianus sp., Ferroplasma sp., Metallosphaera sp., Sulfolobus sp. and Thermoplasma sp.
The method comprises performing a PCR with specific prim ers designed in our laboratories for different taxons SEQID No. 4 to SEQID No.: 407. With qPCR results and other data obtained from the analyzed sample, the microorganism con centration of each analyzed taxon present in the sample is calculated using a mathematical formula.
US 2011/O 1361.25 A1
METHOD FOR THE DENTIFICATION AND QUANTIFICATION OF MICROORGANISMS
USEFUL IN BOMINING PROCESSES
0001. This application is a Continuation-in-Part of U.S. Ser. No. 1 1/509,870, which claims benefit of Serial No. 2179 2005, filed 26 Aug. 2005 in Chile and which applications are incorporated herein by reference. To the extent appropriate, a claim of priority is made to each of the above disclosed applications.
FIELD OF THE INVENTION
0002 The present invention discloses a method to identify and quantify microorganisms useful in biomining processes that are present in a given sample. This method is presented as a useful tool in biomining, in every case where the present microbiological population needs to be evaluated, whether on the mineral, in Solutions, in bioleaching heaps, in biomining laboratories or in any other circumstance that involves the use of Such microorganisms.
BACKGROUND OF THE INVENTION
0003 Biomining is, in general terms, the use of microor ganisms for metal recovery from mineral ores. Its most tra ditional expression is bioleaching, but not only this process is understood as biomining, but also the monitoring and inter vention in Such process, as these techniques are complex and are under constant development; and also laboratory research associated to process improvement or the development of new methodologies. 0004. Until now, bioleaching continues to be the most important process in biomining field, and is defined as a method to solubilize metals from complex matrixes in an acid medium, using direct or indirect microorganism action. The microorganisms that are useful in these processes belong to Bacteria or Archaea kingdoms, and fulfill two basic condi tions: they are acidophilic and chemolithotrophic. 0005 Microbiological Diversity in Communities Associ ated to Bioleaching Processes. 0006 Various microorganisms have been described to be useful in bioleaching processes, and ten taxons could be identified among them: 3 genera and 2 species from the Bac teria kingdom, namely Acidiphilium sp., Leptospirillum sp., Sulfobacillus sp. genera and Acidithiobacillus ferrooxidans and Acidithiobacillus thiooxidans species, and five genera from the Archaea kingdom, namely Acidianus sp., Ferro plasma sp., Metallosphaera sp., Sulfolobus sp. and Thermo plasma sp. (Rawlings D. E. Heavy metal mining using microbes. Annu Rev Microbiol. 2002:56:65-91; Rawlings D E. Characteristics and adaptability of iron- and sulfur-oxidiz ing microorganisms used for the recovery of metals from minerals and their concentrates. Microb Cell Fact. 2005 May 6;4(1):13). 0007 Factors Determining Diversity and Metabolic Activity of the Microbial Community Associated to a Bioleaching Process. 0008 Each of the above mentioned genera or species cata lyzes different reactions and require in its turn different con ditions to perform such reaction, which could be, for instance, aerobic or anaerobic, or could require Some specific nutrient.
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Therefore, the environmental conditions in which a bioleach ing process is performed will modify the bacterial composi tion of the community. 0009. Additionally, the participation of microorganisms in a bioleaching process has been proposed to be direct and/or indirect (Rawlings D. E. Characteristics and adaptability of iron- and Sulfur-oxidizing microorganisms used for the recovery of metals from minerals and their concentrates. Microb Cell Fact. 2005 May 6;4(1):13). When the action is direct, microorganisms directly oxidize the target metal or its counter-ion, in both cases liberating into the solution a target metalion. On the other hand, when the action is indirect, the Substrate of the microorganism is not the target metal neither its counter-ion, but instead chemical conditions are generated that allow the solubilization of said metal, either by acidifi cation of the medium (e.g., by generating Sulfuric acid) or by the generation of an oxidizing agent that ultimately interacts with the salt (metal and counter-ion) to be solubilized. 0010 Regarding this aspect, it is possible that the bacterial community changes its species composition as a function of the bioleaching type being performed in different mineral samples and/or the environmental conditions in which this process is carried out. 0011 For instance, Acidithiobacillus species are able to catalyze the oxidation of reduced Sulfur compounds (e.g., Sulfide, elemental Sulfur, thionates, etc.) using oxygen as electronic acceptor and generating Sulfuric acid as final prod uct and reducing species like Sulfite and thiosulfate as inter mediate products, which allows the solubilization of metals associated to sulfides in the mineral. Acidithiobacillus fer rooxidans and Leptospirillum ferrooxidans are able to cata lyze the oxidation of iron(II) to iron(III) using oxygen as electron acceptor, being the generated iron(III) a great oxi dizing agent that can oxidize sulfides in the mineral or any other compound to be oxidized. 0012. The usual mining practice in bioleaching processes is to leave a mineral heap in an acid medium, generally Sulfuric acid, and constantly remove the acid medium to recover the metal by electrolysis. Usually heaps in which the recovery yield of the metal is efficient are obtained, and also "inefficient' heaps that have a low yield under the same operation conditions and characteristics of the Substrate to be leached. The explanation to this unequal result requires the elucidation of differences in abundance and types of species in the microbiological community between both heaps. In this way, the low yield problem could be explained by the micro bial community composition, and could be solved in its turn by inoculation of microorganisms that catalyze the reaction to be maintained during the bioleaching process. However, a method that enables to quantify the population of archaea and bacteria useful in biomining processes is not available up to this date. 0013. In this patent, a method is described that solves the technical problem previously described, by designing a method to identify and quantify the presence of known micro organisms that are most relevant in biomining processes, namely the bacteria: Acidiphilium sp., Leptospirillum sp., Sulfobacillus sp., Acidithiobacillus ferrooxidans and Acidithiobacillus thiooxidans, and the archaea: Acidianus sp., Ferroplasma sp., Metallosphaera sp., Sulfolobus sp. and Thermoplasma sp. 0014 Nested polymerase chain reaction (PCR) was the technique selected to develop this method. In this technique, a conserved genome region of the microorganisms is firstly
US 2011/O 1361.25 A1
amplified in a first PCR reaction, either on bacteria or archaea. We have selected gene 16SrDNA as the conserved region. Then, taxon-specific primers (targeting genera or species) are used to identify the presence of target microorganisms in a second PCR reaction. This second PCR reaction is performed using an equipment that allows measuring the increase of amplified product in each amplification cycle, and this infor mation allows the quantification, by interpolation, of the original abundance of the target genome in the sample being analyzed. PCR reaction under these conditions is called quan titative PCR or qPCR. 0015. A critical step in nested PCR technique is the design of primers for the second amplification reaction, which have to be specific for the taxon to be determined, and this aspect has a vital importance in this particular case, as the samples to which the process will be applied will usually be metage nomic samples. Therefore, it is necessary to reduce the pos sibility of primer unspecific hybridization to sequences present in the genome of microorganisms that have not yet been identified in the community. We have generated two fundamental tools for the design of these primers: firstly, a depurated 16SrDNA sequence database obtained from all disclosed 16SrDNA sequences; and a computational program for primer design that uses as input such database and allows designing thermodynamically stable taxon specific primers. 0016. In the state of the art there are many examples of the application of nested PCR or qPCR, but none of them is focused to bacteria or archaea useful in biomining processes. For instance, J. L. M. Rodrigues et al (Journal of Microbio logical Methods 51 (2002) 181-189) describe a qPCR to detect and quantify PCB-degrading Rhodococcus present in soil, where the 16SrDNA gene belonging to the strain with the target activity is sequenced, specific primers for said sequence are designed and qPCR reactions are carried out using said primers. In this document, a direct qPCR approach is used, instead of a nested qPCR, and it is directed to other type of microorganisms, whose handling has been widely studied and many techniques for DNA extraction are avail able. Another document that uses a similar approach is Patent Application EP 1484 416, which discloses a method for the detection and quantification of pathogen bacteria and fungi present in an environment sample using qPCR. The method comprises the extraction of DNA from bacteria and fungi present in an environment sample, obtaining specific sense and antisense primers for each of the taxons to be detected and quantified; and performing qPCR reactions using a pair of primers for each of the target pathogens. 0017 Although it is possible to enumerate documents in which microorganisms are identified and quantified using quantitative PCR techniques, as they are well known tech niques in the art, the relevant point is the generation of a depurated database that allows to design specific primers and has not been implemented before for the identification of microorganisms useful in biomining processes, which is Sub ject matter of this invention.
DETAILED DESCRIPTION OF THE INVENTION
0018. As has been anticipated, the invention relates to a method that allows the identification and quantification of essential microorganisms in biomining processes. These essential microorganisms belong to 10 taxons, the genera Acidiphilium sp., Leptospirillum sp., Sulfobacillus sp. and the species Acidithiobacillus ferrooxidans and Acidithiobacillus thiooxidans belonging to the Bacteria kingdom; and the gen
Jun. 9, 2011
era Acidianus sp., Ferroplasma sp. Metallosphaera sp., Sul folobus sp. and Thermoplasma sp. belonging to the Archaea kingdom. 0019. As previously indicated, a method to identify and quantify biomining microorganisms would have applications in different industrial tasks and areas. For instance, a good tool for suitable control of bioleaching process could be the identification of microorganisms that are present in a bioleaching heap and how abundant they are, as it could be established whether is necessary to inoculate some particular microorganism or simply determine which nutrients should be added to the mixture, thus maximizing the quantity of mineral recovered in the process. The idea is to correlate the recovery efficiency of different metals present in the heap with the composition of the microbiological community in the heap, referred to the number and type of present individu als. 0020. In general terms, samples to be analyzed in the method of the invention will be biomining samples, but this does not limit the scope of the invention, as the described method could be applied any time that one or more of the 10 taxons Subject of this invention is to be identified and quan tified. 0021. In the description of the invention, all oligonucle otide sequences are written in direction 5' to 3". Described oligonucleotides correspond to primers for PCR reactions, which can be sense or antisense primers, which could be indicated specifically (e.g., as table titles) or alternatively by including letter “F” for sense or forward primers and “R” for antisense or reverse primers in the name of the primer. 0022. The following is the description of each of the stages of the method in detail. (0023 DNA Preparation. 0024. In a first stage, it is necessary to extract DNA from the sample. Different methods to extract DNA from mineral or soil samples have been disclosed and any of them can be used, considering in each case the particular nature of the sample (Appl Environ Microbiol. 2003 July; 69(7):4183-9: Biotechniques. 2005 April: 38(4):579-86). In the case that total extracted DNA (from mineral samples, being e.g. grounded chalcopyrite type 1 or other) is turbid or has a yellow or orange color, it is recommended to re-purify the sample using any existing purification technique; in our labo ratories this step is performed using commercial DNA puri fication columns. The purified fraction is resuspended inster ile nuclease-free H.O. 0025. Once total DNA samples have been purified, total DNA present in the sample should be quantified; again, this quantification could be performed using any existing method to quantify DNA; in our laboratories it is done by spectro photometry. 0026. After quantifying total DNA present in the sample, an aliquot is taken and diluted to a concentration Suitable for the method, which finally ranges from 0.5 to 40 ng/ul, pref erably from 1 to 30 ng/ul, and most preferably from 1 to 10 ngful. The dilution must be done using sterile nuclease-free Water.
0027 All determinations of biomining microorganisms presented here are based in a specific recognition of a frag ment of 16S rDNA genes being unique for the taxon under analysis and not presenting any cross reaction with other microorganisms. The method claims the protection of several oligonucleotide primers that specifically amplify a fragment of 16S rDNA being used as identification tag. This method is
US 2011/O 1361.25 A1
even useful when analyzing complex samples with DNA coming from different microorganisms. The method com prises a standard curve construction, a qPCR reaction with the designed specific primers and the final data transformation. 0028. The standard curve is constructed using the specific PCR product fragment of the 16S rDNA gene produced by the designed primers in different dilutions of a mixed DNA stan dard sample. 0029 PCR Reaction. 0030 A plurality of PCR is carried out, specific for each taxon to be identified, using specific primers that amplify inside the 16S rDNA region. 0031. In this stage it is crucial to have specific and efficient primers to amplify the target fragment that have no cross reaction with organisms from other taxons and are thermo dynamically stable, i.e. do not form hairpins, homodimers or heterodimers. The primers used in this application have been designed using the method disclosed in Patent Application CL 2102-2005 filled by Biosigma; as said method guarantees the efficiency and specificity of the designed primers. 0032. PCR will be performed on all the reaction products. Advantageously, all reactions are carried out in duplicate, and a negative control is added. 0033. It is important to point out that the method of the invention can be carried out to identify and quantify either all the described taxons or only one of them, and also all the possible intermediate combinations, and as a consequence every one of these options will remain being comprised inside the scope of the present invention. 0034. The PCR is a quantitative PCR (qPCR), therefore it should be performed in a suitable thermocycler and using fluorescent reagents for qPCR. There are different commer
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cially available alternatives, either for equipment or reagents, and any of them can be selected to carry out the present method. 0035. For the PCR reaction the following mix is prepared:
TABLE 4
Sterile nuclease-free H2O 10.5 ul Sense primer (10M) 0.5 ul Antisense primer (10M) 0.5 ul qPCR reagent 12.5 ul
To the mix described in Table 4, 1 ul of DNA or sterile water for the qPCR blank is added.
Primers for the PCR
0036. As previously indicated, the requirements to be full filled by each primer pair selected for the PCR are: being specific for each taxon, having no cross-reactivity and being thermodynamically stable to assure primer availability in the PCR reaction. Our laboratory has developed a primer design program that gives a large amount of primers fulfilling these requirements. The method of the invention can be performed by combining any sense primer with any antisense primer designed by our program. In following tables, we give 20 sense primers and 20 antisense primers for each taxon, where any possible combination thereof could be selected for the qPCR. (Note: the sequences of the designed primers have been com pared, by using Blast from NCBI, with previously existent sequence disclosures, thus guaranteeing its novelty as prim ers.) 0037 Bacteria Kingdom:
TABLE 5
Acidiphilium sp.
Sense primers Seq ID Nos. Antisense primers Seq ID Nos.
CAA CCA CGG TCG GGT CAG A (SEQ
GAC CTT AAG TTG ATG CGC T (SEQ
AGT CAA CCA CGG TCG GGT C (SEQ
GGT TTG ACC TTA AGT TGA (SEQ TG
CTT. AAG TTG ATG CGC TAA C (SEQ
GGC AGT CAA CCA CGG TCG G (SEQ
CGA TGC TGA GCT GAT CCT G (SEQ
AAG TTG ATG CGC TAA CCG C (SEQ
AAA GTC GCC TAA GGA. GGA G (SEQ
GTC GCC TAA GGA GGA GCC T (SEQ
AAG GAG GAG CCT GCG TCT G (SEQ
AGG AGC CTG CGT CTG ATT A (SEQ
AGG AGG CAG TCA ACC ACG (SEQ GT
GCG AAA GTC GCC TAA GGA G (SEQ
D NO: 4) TCT CTG ACC CGA CCG TGG TT (SEO ID NO. 24)
D NO; 5) TCA ACT TAA GGT CAA ACC AA (SEO ID NO: 25)
D NO : 6) GGA GCT TAT TCT GCG GGT A (SEO ID NO: 26)
D NO: 7) GCA TCA ACT TAA GGT CAA AC (SEO ID NO: 27)
D NO: 8) AGC GCA TCA ACT TAA GGT CA (SEO ID NO: 28)
D NO: 9) GTT AGC GCA TCA ACT TAA GG (SEO ID NO : 29)
D NO : 1.O.) CCG ACC GTG GTT GAC TGC C (SEO ID NO : 3O)
D NO : 11) GGA TCA GCT CAG CAT CGC TG (SEO ID NO : 31)
D NO : 12) TCA GGA TCA GCT CAG CAT CG (SEO ID NO : 32)
D NO : 13) CGG TTA. GCG CAT CAA CTTA (SEO ID NO : 33)
D NO : 14) GGC. TCC TCC TTA. GGC GAC TT (SEO ID NO. 34)
D NO : 15) GTT GAC TGC CTC CTT GCG GT (SEO ID NO : 35)
D NO : 16) TCC TCC TTA. GGC GAC TTT CG (SEO ID NO : 36)
D NO: 17) GTG GTT GAC TGC CTC CTT GC (SEQ ID NO : 37)
US 2011/O 1361.25 A1 Jun. 9, 2011 10
TABLE 14 - continued
Thermoplasma sp.
Sense primers Seq ID Nos. Anti sense primers Seq ID Nos.
TAA CTC GCC CTC. ACG AAT GT (SEO ID NO : 378) TTA. CAG CCC TCA GTC CTG ACT (SEO ID NO: 398)
GAA. GGT GTT AAG TGG GTC A (SEO ID NO : 379) AAT CCA CAT TCG TGA GGG CGA (SEO ID NO: 399)
AAA CCC GTT CGT AGT CAG GAC (SEO ID NO: 38O) ATG GGG GTC TTG CTC GTT AT (SEQ ID NO: 4 OO)
TAC GGT GAA. TAT GCC CCT GC (SEO ID NO: 381) GCT GTT GAC CTA CGA TTA. C (SEQ ID NO: 401)
CAC TTG GTG TTG CTT CTC CGT (SEO ID NO: 382) CCT ACG ATT ACT ACG GAA TCC (SEO ID NO. 4 O2)
GAT CAC TTT TAT TGA GTC T (SEO ID NO: 383) ACC CAC TTA ACA CCT TCG C (SEQ ID NO: 4 O3)
AGC ATC AGG AAT AAG GGC TG (SEO ID NO: 384) CCC AAG TCT TAC AGT CTC TT (SEQ ID NO: 4O4)
AAG ACC CCC ATC. TCT AAT T (SEO ID NO: 385) CTA CCC TGA AAG TAC ATT CA (SEQ ID NO: 405)
CCG GTC TTA TAA ATC TTC A (SEO ID NO: 386). CAG CCC TTA TTC CTG ATG C (SEQ ID NO: 406)
ATA ACG. AGC AAG ACC CCC AT (SEO ID NO: 387). GGT CGT CCT TTC AGG ATT AC (SEO ID NO: 4 O7)
In PCRs a reaction is also included to quantify total bacteria or archaea present in the sample; in this case known universal TABLE 1.5 - continued primers are used for both kingdoms which are selected among
PCR the primers included in Table 15.
Seq ID Nos. TABLE 1.5
Univ534-R ATT ACC GCG GCT (SEO ID NO: 417) PCR GCT GG
Seq ID Nos.
Bacteria primers Each PCR has a specific cycle, wherein the alignment ten perature changes, said temperature being specific for each
Eub27 F AGA GTT TGA TCC (SEO ID NO : 1) used primer pair. Table 16 Summarizes general conditions for TGG CTC AG all qPCR cycles.
Univ533-F" GTG CCA GCM GCC (SEO ID NO : 408) GCG GTA TABLE 16
Bact358-F CCT ACG GGA. GGC (SEQ ID NO: 409) Step Temperature (C.) Time (s) AGC AG 1 Initial denaturation 95 120
2 Denaturation 95 30 Univeo 7-R. CCG TCA ATT CCT (SEO ID NO : 41O) 3 Alignment (*) 30
TTG. AGT T 4 Extension 72 40 5 Pre-reading 8O 10
Bact338-R GCT GCC TCC CGT (SEO ID NO : 411) 6 Reading 8O AGG AGT Repeat 40 times from step 2 to step 6 (qPCR cycle)
7 Denaturation curve Between 70 and 100° C., reading each Bact1387-R. GGG CGG WGT GTA (SEQ ID NO: 412) O.2°C.
CAA. GGC () specific temperature for each used primer pair
Archaea primers Denaturation curve carried out at the end of cycle 40, gives the
Arch344-F ACG GGG CGC AGC (SEQ ID NO: 413) Tm of the amplification product, and is also used to establish AGG CGC GA whether more than one amplification product is present in the
amplified sample, as each would generate its own curve. 7 Univs1.5-F GTG CCA GCA GCC (SEQ ID NO: 414) 0039. The qPCR thermocycler gives a result correspond
GCG GTA A ing to DNA concentration present in each reaction, and this Arch958-R YCC GGC GTT GAM (SEO ID NO : 415) information 1s used to calculate the number of microorgan
TCC AAT T sms present in the sample, which is called Q. This value is inferred by the computational program associated to the ther
Arch915-R' GTG CTC CCC CGC (SEO ID NO : 416) mocycler based on: DNA concentration in calibration curve CAA TTC CT reactions and the cycle in which sample begins to amplify (or
to exponentially increase its fluorescence value). The corre
US 2011/O 1361.25 A1
lation between the logarithm of DNA concentration and the cycle in which amplification is observed generates a linear equation, from which DNA concentration in the analyzed samples is inferred. 0040 Calculation of the Number of Microorganisms Present in the Sample. 0041 Taking into account the qPCR result and other data generated during the process, the inventors have developed a mathematical formula that allows calculating the exact num ber of microorganisms from a given taxon present in a given sample, specially a biomining sample. 0042. By applying the method of the invention, the num ber of microorganisms belonging to the taxons Acidiphilium
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sp., Leptospirillum sp., Sulfobacillus sp. Acidithiobacillus ferrooxidans and Acidithiobacillus thiooxidans, Acidianus sp., Ferroplasma sp., Sulfolobus sp., Metallosphaera sp., and/ or Thermoplasma sp. present in a sample can be determined. 0043. The method comprises a standard curve construc tion, a qPCR reaction with the designed specific primers and the final data transformation. 0044) The standard curve is constructed using the specific PCR product fragment of the 16S rDNA gene produced by the designed primers in different dilutions of a mixed DNA stan dard sample. 0045. The standard curve obtained with the dilutions of the standard DNA mixture will have a profile as the follow
US 2011/O 1361.25 A1
Cd s
a. s
c s H ''
d
30
28
26 -
24
22 -
2O -
18
16
14 -
12
is s k k
12
y = 1.0017x - 0.0398
15
C(T) para Est. Antiguos
25 30
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Standard DNA mixture Diution 1:10 Dilution 1:100 Dilution 1:1 OOO Dilution 1:10000 Dilution 1:1 OOOOO
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Whereas C(T) is the value given by the equipment that is related to the DNA concentration of the samples used with known concentration (different dilutions) of the same taxon. One standard curve has to be made for each taxon to be analyzed. 0046. Using this information and the following equation it
is possible to correlate the concentration of DNA with the number of molecules of 16S rRNA and therefore microor ganisms of specific taxon. The standard curve can contain a mixture of DNAs coming from different species but even in this case one can be able to determine the number of mol ecules of specific specie knowing the length of the PCR product to be amplified by the specific primers and the con centration of initial DNA as it was added for the standard CUV.
6.023x105unlock Angul (1) B: 660ginot : 1 x 10, QN of molecules F
Whereas
0047 1 bp=660 g/mol I0048 6.023x10' molecules or copies (Avogadro num
ber) 0049 A-DNA concentration in ng/uL 0050 B-Length of the PCR product
0051. As only A and B are variables, formula can be expressed as:
(Anglu) (2) One of molecules = 9.126x10cing: inolecing
For problem samples the qPCR reaction is performed accord ing to the best conditions recommended for the PCR product to be amplify and the result obtained as a C(T) value is then interpolated in the standard curve to obtain the concentration of specific DNA fragment tag present in the sample. With the concentration and equation (2) one can obtain the number of molecules present in a sample as determined by the qPCR reaction. Finally to estimate microorganisms concentration the follow ing equation must be applied:
N QN molecules *Tng (3) Ceutni) = . . . . . Cmolecules/cel * Ung * Cn(mL)-(g)
0.052 N=Estimated Concentration of microorganisms. 0053 Q-Number of Molecules as calculated with the standard curve using the C(T) value given by the equip ment.
0054 T=Total DNA isolated from original sample. 0055 U=Amount of DNA used in the qPCR reaction. 0056 C=Number of molecules or copies by genome of the specific DNA fragment detected.
0057 Cm=Amount in “g” or “ml” of the processed sample.
0058. In all cases number of molecules present by genome's microorganisms (C) is known either by sequence available publicly or Southern blot performed in our lab. For
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example, in the case of A. ferrooxidans it is known that the specie DSM 16786 Wenelen has only one copy of 16S rDNA gene but in the case of Leptospirillum DSM 17947Yagan it is reported to have 2 copies of the 16S rDNA gene.
EXAMPLES
Example 1
Quantification of Acidithiobacillus thiooxidans, Acidithiobacillus ferrooxidans, Leptospirillum sp. and Acidiphilium sp. Present in a Biomining Sample
0059. Two solid samples obtained from mineral bioleach ing heaps (SS-1 and SS-2) and 2 liquid samples recovered from bioleaching effluents (LS-1 and LS-2) were analyzed and total DNA was extracted from each sample. For all solid samples a further step was necessary, a re-puri fication of DNA, which consisted in a sample re-purification using any existing purification technique; in our laboratories this step is performed using commercial DNA purification columns to obtain a translucent appearance in the extraction Solution.
Total DNA was quantified in each sample using a NanoDrop 1.0 spectrophotometer. Total extracted DNA nanograms (T) are shown in Table 1-1 together with the initial sample vol umes (C). Registered results were:
TABLE 1-1
Sample T Cn
SS-1 543.2 100 g
SS-2 1660.4 20 g
LS-1 11365.2 45 mL.
LS-2 16364.6 45 mL.
Each of these samples was diluted with sterile nuclease-free water in order to obtain a concentration in the range 0.5 to 30 ng/ul. Table 1-2 shows the final volume to which the DNA Solution was brought and its final concentration.
TABLE 1-2
Sample Final volume (l) Concentration (ngful)
SS-1 70 7.76
SS-2 70 23.72
LS-1 421 27
LS-2 545.5 30
A calibration curve was simultaneously prepared for each taxon to allow the calculation of molecule number of the target gene in experimental samples. The standard DNA was prepared for each taxon using PCR products obtained with specific primers shown in table 1-3.
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TABLE 1-3
Microorganism to be Alignment determined temperature Used primers
Total bacteria GCA GCC GCG GTA-3
ATT CCT TTG. AGT T-3'
A. ferrooxidans 6 Oc C. (P.13) F: GGG AGA. GGG TG-3
(P. 6) R: ATT CCG GAC AT-3'
A. thiooxidans 6 o C. (P. 7) F: TGC TGT TGA C-3'
(P.11) R: AGA CCT AAT G-3
Leptospirillum sp. 588 C. (P. 4) F: AAG GGT TTC G-3
(P. 2) R: GGG ATA TTT A-3
Acidiphilium sp. 618 C. (P.10) F: GGA. GGA, GCC T-3'
(P. 3) R: TCT GCG GGT A-3'
The PCR reaction to obtain the PCR products was carried out using as substrate the respective genomic DNA (200 ng) from each of the following microorganisms: Acidithiobacillus thiooxidans, Acidithiobacillus ferrooxidans, Leptospirillum sp. and Acidiphilium sp. The composition of PCR mix is detailed in table 1-4. The PCR product was purified using commercial DNA purification columns and concentration of the respective PCR products was determinate using Nano Drop 1.0 spectrophotometer.
TABLE 1-4
Reagent 1 reaction
Sterile nuclease-free H2O 18.35 ul PCR Buffer 10x 2.5 ul MgCl2 (50 mM) 1.5 ul dNTPs (10 mM each) 0.5 ul Primer Forward* (10 M) 0.5 ul Primer Reverse (10M) 0.5 ul Hot Start Taq (5 U?ul) 0.15 ul
*Used primers are described in Table 1-3.
More specifically, the PCR products were obtained using genomic DNA from the following strains:
0060 A. ferrooxidans DSM 16786; 0061 A. thiooxidans DSM 504; 0062 Leptospirillum sp. DSM 1931 and 0063 Acidiphilium acidophilus DSMZ 700.
The number of molecules for each PCR product was calcu lated using the equation (2) described above. Each standard curves was prepared using five serial dilutions containing 1x10 molecules of specific PCR products in a final volume of 20 ul, which is included in the calibration CUV.
6 o C. (P.1) 533-F: 5' - GTG CCA
(P. 2) 907-R: 5' - CCG TCA
5 - CTA GAG TAT
s' - CTC TGC AGA
s' - TAA TAT CGCC
s" - TTT CAC GAC
5 - GGA ACC GTG
s' - CCG TCA TCG
s' - GTC GCC TAA
5 - GGA GCT TAT
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Amplify lengths (bp)
374
365
146
315
263
0064. A reaction “master mix' for the quantitative PCR was prepared wherein the amount of each constituent was multiplied by the total number of reactions to be carried out in order to homogenize reagent concentrations in the different PCR tubes. The reaction mix was aliquoted in 0.2 ml tubes, using a Volume of 24 Jul of reaction mix per tube. Subsequently 5 quantitative PCR were performed, one for each taxon: Acidithiobacillus thiooxidans, Acidithiobacillus ferrooxidans, Leptospirillum sp., Acidiphilium sp. and one for total bacteria, using specific primers for each of them. Sense and antisense primers were selected for the different taxons from those designed specifically by our method. Primers used for each taxon and their respective annealing temperatures are indicated in Table 1-3.
In the present Example, the following reactions were per formed in duplicate:
0065 0.066 b) Five reactions for each point of calibration curve (n-6), corresponding to standard DNA mastermix concentrations of 1x10, 1x10", 1x10, 1x10 and 1x10" molecules, and a blank.
0067. One quantitative PCR reaction was carried out on each sample and on standard DNA, these reactions being performed in duplicate. The qPCR was carried out using Mix SYBR Green qPCR. Each quantitative PCR reaction was carried out by duplicate which gives a total of 40 reactions per each taxon.
a) Five reaction for each sample (n=4) and
The quantitative PCR reaction mix for each taxon is shown in Table 1-5, where primers used are those described on Table 1-3.
US 2011/O 1361.25 A1
TABLE 1-5
1 reaction 20 reactions
Sterile nuclease-free H2O 16.1 ul 322 Jul Primer 1 (10 M) 0.5 ul 10 ul Primer 2 (10 M) 0.5 ul 10 ul PCR Buffer 10x 2.5 ul 50 ul MgCl2 (50 mM) 1.5 ul 30 ul dNTPs (10 mM each) 2.5 ul 50 ul Hot Start Taq (5 U?ul) 0.15 ul 3 ul SYBR Green qPCR 100x 0.25 ul 5 ul
This reaction mix was homogenized and aliquoted in 200.2 ml tubes, which were duly labeled. To each of the tubes 1 ul of DNA sample dilution or 1 ul of sterile nuclease-free water for the blank was added. PCR tubes containing the reaction mix and sample were shaked at Vortex for 5 seconds and centrifuged for 1 minute at 2000xg, in order to homogenize and bring the reaction liquid to the bottom of the tube, respectively. Then, the tubes with quantitative PCR reactions were sub jected to temperature cycles for amplification (table 1-6). According to the microorganism to be determined, different primer pairs were used and therefore different amplification programs were used. In the following Table, amplification programs used in the different PCR reactions are shown.
TABLE 1-6
Step Temperature (C.) Time (s)
1 Initial denaturation 95 120 2 Denaturation 95 30 3 Alignment (*) 30 4 Extension 72 40 5 Pre-reading 8O 10 6 Reading 8O
Repeat 40 times from step 2 to step 6 (qPCR cycle) 7 Denaturation curve Range between 70 and 100° C., reading
each 0.2°C.
() specific temperature for each used primer pair, as indicated in Table 1-3,
When the quantitative PCR is finished, all data generated by the qPCR thermocycler are stored, from standard curve is calculated the number of molecules for target gene into each sample; this data corresponds to Q and is shown in Table 1-7. wherein DNA amounts in nanograms used for each reaction are included (U).
Jun. 9, 2011 15
0068 Calculation of the Number of Microorganisms Present in the Samples. 0069. Taking into account the qPCR result and data gen erated during the process, the following formula described as equation (3) can be applied:
Noel (mL)-(g) = QN molecules *Tng unt - . Cmolecules/cel * Ung * Cn(mL)-(g)
0070 N=Estimated Concentration of microorganisms. (0071 Q-Number of Molecules as calculated with the
standard curve using the C(T) value given by the equip ment.
0.072 T-Total DNA isolated from original sample. 0.073 U=Amount of DNA used in the qPCR reaction. 0.074 C=Number of molecules or copies by genome of the specific DNA fragment detected. (see table 1-8)
0075 Cm=Amount in or “ml of the processed sample
9.
TABLE 1-8
Number of copies of 16S rDNA gene in the genome of different taxons as described in literature or determined by Southern blot.
Microorganisms C
A. ferrooxidans A. thiooxidans Acidiphillium Leptospirillaim Bacteria total
*The number of 16S rDNA copies by genome of total bacteria was estimated as an average according to our observations,
According to the previous, the following microbiological populations were determined in the analyzed samples:
TABLE 1-9
SS-1
Bacteria Mo.g. of sample
Total bacteria S.27E--OS A. ferrooxidans 1.OOE--O3 A. thiooxidans 1.29E-04 Leptospirilium sp. S.2SE--O2 Acidiphilium sp. 4.94E--04
TABLE 1-7
Q
Total Leptospirillum Acidiphilium
Sample bacteria A. ferrooxidans A. thiooxidans sp. Sp. U
SS-1 4.85E-05 O.OOSE-OS O.06E--OS O.OOSE-OS O4SE--OS 2.5
SS-2 3.48E--OS O.O47E--OS O.O13E--OS O.71E--OS O.34E--OS 2.5
LS-1 3.12E--OS O.SSE--OS O O.OO6E--OS O 2.5
LS-2 2.88E--OS O.82E--OS O O.OO6E--OS O 2.5
US 2011/O 1361.25 A1
TABLE 1-10
SS-2
Bacteria Mo.g. of sample
Total bacteria 5.78E--06
A. ferrooxidans 1.SSE--OS A. thiooxidans 4.15E-04
Leptospirilium sp. 1.17E--O6 Acidiphilium sp. S.71E--OS
TABLE 1-11
LS-1
Bacteria Mo./ml of sample
Total bacteria 1.57E--O7 A. ferrooxidans 5.61E--O6 A. thiooxidans O.OOE--OO Leptospirilium sp. 3.02E--04 Acidiphilium sp. O.OOE--OO
TABLE 1-12
LS-2
Bacteria Mo./ml of sample
Total bacteria 2.09E-07 A. ferrooxidans 1.19E--O7 A. thiooxidans O.OOE--OO Leptospirilium sp. 4.77E--04 Acidiphilium sp. O.OOE--OO
Microorganism to be determined
Total bacteria
Sulfobacillus sp.
Total archaea
Sulfolobus sp.
16
temperature Used primers
Jun. 9, 2011
Example 2 Quantification of Sulfobacillus sp., Sulfolobus sp.,
and Ferroplasma sp. in a Sample 0076 One sample obtained from mineral bioleaching heap (SS-3) and one liquid sample recovered from bioleach ing effluents (LS-3) were analyzed and total DNA was extracted from each one. A further DNA re-purification step was required to obtain a translucent appearance in the extraction solution. Total DNA was quantified in each sample using a NanoDrop 1.0 spectrophotometer. Total extracted DNA nanograms (T) are shown in Table 2-1 together with the initial sample vol umes (C).
TABLE 2-1
Sample T Cn
SS-3 537.25 20 g LS-3 1621.13 50 mL.
Each of these samples was diluted with sterile nuclease-free water in order to obtain a concentration in the range of 0.5 to 30 ng/ul. Table 2-2 shows the final volume to which the DNA Solution was brought and its final concentration.
TABLE 2-2
Sample Final volume (l) Concentration (ngful)
SS-3 70 7.67 LS-3 540 30
Five calibration curves were prepared for quantitative PCR that includes total Bacteria, Sulfobacillus, total Archaea, Fer roplasma and Sulfolobus. These calibration curves were used for calculating the molecule number of target gene in experi mental samples. The standard DNA was prepared for each taxon using PCR products obtained with specific primers described in table 2-3.
TABLE 2-3
Alignment Amplify lengths (bp)
59 (P. 1) 27-F: 5'-AGA GTT TGA 374 TCC TGG CTC AG-3'
(P. 2) 338-R 5 " - GCT GCC TCC CGT AGG AGT-3'
66 (P.1) F : 5'- CGA AGG CGG TGC 122 ACT GGC C-3
(P. 3) R: 5'-GGT GGA CCC CCG CGA CAC C-3
6 O (P.1) 515-F: 5' - GTG CCA GCA 443 GCC GCG GTA A-3
(P. 2) 958-R: 5'-TCC GGC GTT GAA TCC AAT T-3'
6 O (P. 3) F : 5' - GTC CTG GAA CGG 282 TTC CTC G-3
(P.9) R. 5" - CCC CCG CCT TTG TAG AGC G-3
US 2011/O 1361.25 A1
TABLE 2-3 - continued
Jun. 9, 2011
Microorganism to be Alignment Amplify determined temperature Used primers lengths (bp)
Ferroplasma sp. 56 (P.17) F : 5'-TGG CCA AGA CTT 2O4. TTC TCA T-3
(P.19) R: CTT GCA GT-3'
The PCR reaction to obtain the PCR products was carried out using the respective primers and as Substrate the respective genomic DNA (200 ng) from each of the following microor ganisms: Sulfobacillus, Ferroplasma y Sulfolobus. Substrate for PCR products for standard representing Total Archaea was obtained from the mix of the genomic DNA containing 100ng of Ferroplasma and 100 ng of Sulfolobus. Substrate for PCR product representing Total Bacteria was obtained using DNA from Sulfobacillus (200 ng). The composition of PCR mix is detailed in table 2-4 and PCR amplification program in table 2-5. The respective PCR products were purified using commercial DNA purification columns and concentration of them was determinate using NanoDrop 1.0 spectrophotom eter.
TABLE 2-4
Reagent 1 reaction
Sterile nuclease-free H2O 18.35 ul PCR Buffer 10x 2.5 ul MgCl2 (50 mM) 1.5 ul dNTPs (10 mM each) 0.5 ul Primer Forward* (10 M) 0.5 ul Primer Reverse (10M) 0.5 ul Hot Start Taq (5 U?ul) 0.15 ul
*Used primers are described in Table 2-3.
TABLE 2-5
Temperature Step (° C.) Time (s)
1. Initial denaturation 95 120 2. Denaturation 95 30 3. Alignment 30 4. Extension 72 120
Wherein steps 2 to 4 are to be repeated 28 times, The alignment temperature used is according to pairs of primer detailed in table 2-3,
The number of molecules for each PCR product was calcu lated using the equation (2) described above. Each standard curves was prepared using five serial dilutions that containing 1x10 molecules of specific PCR products in a final volume of 20 ul, which in its turn is included in the calibration curve.
0077. A reaction “master mix” for the quantitative PCR was prepared wherein the amount of each constituent was multiplied by the total number of reactions to be carried out in order to homogenize reagent concentrations in the different
s' - CCG ATC. TCA TGT
PCR tubes. The reaction mix was aliquoted in 0.2 ml tubes, using a Volume of 24 Jul of reaction mix per tube. Subsequently 5 quantitative PCR were performed, one for each taxon: Sulfobacillus, Ferroplasma, Sulfolobus, Total Bacteria and one for Total Archaea, using specific primers for each of them. Sense and antisense primers were selected for the different genera from those included in the description of the tables corresponding to each taxon. Primers used for each taxon and their respective annealing temperatures are indi cated in Table 2-3.
In the present Example, the following reactions were per formed in duplicate:
0078 0079 b) Five reactions for each point of calibration curve (n-6), corresponding to standard DNA master mix concentrations of 1x10, 1x10", 1x10, 1x10 and 1x10" molecules, and a blank.
One quantitative PCR reaction was carried out on each sample and on standard DNA, these reactions being per formed in duplicate. The qPCR was carried out using Mix SYBR Green qPCR. Each quantitative PCR reaction was carried out by duplicate which gives a total of 16 reactions per each taxon.
a) Five reaction for each sample (n=2) and
The quantitative PCR reaction mix for each taxon is shown in Table 2-6, where primers are those that are corresponding according to Table 2-3.
TABLE 2-6
1 reaction 16 reactions
Sterile nuclease-free H2O 16.1 ul 257.6 ul Primer 1 (10 M) 0.5 ul 8 ul Primer 2 (10 M) 0.5 ul 8 ul PCR Buffer 10x 2.5 ul 40 ul MgCl2 (50 mM) 1.5 ul 24 ul dNTPs (10 mM each) 2.5 ul 40 ul Hot Start Taq (5 U?ul) 0.15 ul 2.4 ul SYBR Green qPCR 100x 0.25 ul 4 Jul
This reaction mix was homogenized and aliquoted in 200.2 ml tubes, which were duly labeled. To each of the tubes 1 ul of DNA sample dilution or 1 ul of sterile nuclease-free water for the blank was added.
PCR tubes containing the reaction mix and sample were shaked at Vortex for 5 seconds and centrifuged for 1 minute at 2000xg, in order to homogenize and bring the reaction liquid to the bottom of the tube, respectively. Then, the tubes with quantitative PCR reactions were sub jected to temperature cycles for amplification (table 2-7).
US 2011/O 1361.25 A1 18
According to the microorganism to be determined, different primer pairs were used and therefore different amplification programs were used. In the following Table, amplification programs used in the different PCR reactions are shown.
TABLE 2-7
Jun. 9, 2011
TABLE 2-7-continued
Step Temperature (C.) Time (s)
5 Pre-reading 8O 10 6 Reading 8O
Repeat 40 times from step 2 to step 6 (qPCR cycle) 7 Denaturation curve Between 70 and 100° C.,
reading each 0.2°C. Step Temperature (C.) Time (s) () specific temperature for each used primer pair, as indicated in Table 2-3.
1 Initial denaturation 95 120 When the quantitative PCR is finished, all data generated by 2 Denaturation 95 30 the qPCR thermocycler are stored, from standard curve is 3 Alignment (*) 30 calculated the number of molecules for target gene into each
sample; this data corresponds to Q and is shown in Table 2-8. 4 Extension 72 40 wherein DNA amounts in nanograms used for each reaction are included (U).
TABLE 2-8
-O-
Total Sulfolobus Ferroplasma Sample Total bacteria Sulfobacilius sp. archaea sp. sp U
SS-3 S.76E--OS O.14E--OS 1.61E--OS O O.O28E--OS 2.5 LS-3 16.4E--05 O.O18E--OS 16.8E--OS 0.27E--OS 4.7SE-OS 2
0080 Calculation of the Number of Microorganisms Present in the Sample. 0081. The standard curve obtained with the dilutions of the standard DNA mixture will have a profile as the follow ing:
US 2011/O 1361.25 A1 Jun. 9, 2011
| Standard DNA mixture y = 1.0017x - 0.0398 Diution 1:10
R2 = 1 Dilution 1:100 Dilution 1:1000 Dilution 1:1 OOOO Dilution 1:1 OOOOO
d > s 2
s c as
Whereas C(T) is the value given by the equipment that is related to the DNA ConCentration of the samples used with known
2O concentration (different dilutions) Of the Same taXOn. One Standard Curve has to be made for each taxon to be analyzed.
C(T) para Est. Antiguos
US 2011/O 1361.25 A1
0082. Using this information and the following equation it is possible to correlate the concentration of DNA with the number of molecules of 16S rRNA and therefore microor ganisms of specific taxon. The standard curve can contain a mixture of DNAs coming from different species but even in this case one can be able to determine the number of mol ecules of specific specie knowing the length of the PCR product to be amplified by the specific primers and the con centration of initial DNA as it was added for the standard CUV.
6.023x105unlock Angul (4) B: 660ginot : 1 x 10, QN of molecules F
Whereas
I0083. 1 bp=660 g/mol 10084) 6.023x10' molecules or copies (Avogadro num
ber) I0085 A-DNA concentration in ng/uL I0086 B-Length of the PCR product
0087 As only A and B are variables, formula can be expressed as:
(Anglu) (5) QNo of molecules = 9.126X 1Ol inolecing
For problem samples the qPCR reaction is performed accord ing to the best conditions recommended for the PCR product to be amplify and the result obtained as a C(T) value is then interpolated in the standard curve to obtain the concentration of specific DNA fragment tag present in the sample. With the concentration and equation (2) one can obtain the number of molecules present in a sample as determined by the qPCR reaction. Finally to estimate microorganisms concentration the follow ing equation must be applied:
N QN molecules *Tng (6) Ceit (mL), (g) Cmolecules/cel * Ung * Cn(mL)(g)
I0088 N=Estimated Concentration of microorganisms. I0089 Q-Number of Molecules as calculated with the
standard curve using the C(T) value given by the equip ment.
(0090 T=Total DNA isolated from original sample. (0091 U=Amount of DNA used in the qPCR reaction. 0092 C=Number of molecules or copies by genome of the specific DNA fragment detected.
(0093. Cm=Amount in “g” or “ml” of the processed sample.
Microorganism to be determined
Total bacteria
20 Jun. 9, 2011
0094. In all cases number of molecules present by genome's microorganisms (C) is known either by sequence available publicly or Southern blot performed in our lab. For example, in the case of A. ferrooxidans it is known that the specie DSM 16786 Wenelen has only one copy of 16S rDNA gene but in the case of Leptospirillum DSM 17947Yagan it is reported to have 2 copies of the 16S rDNA gene.
EXAMPLES
Example 1
Quantification of Acidithiobacillus thiooxidans, Acidithiobacillus ferrooxidans, Leptospirillum sp. and Acidiphilium sp. Present in a Biomining Sample
0095. Two solid samples obtained from mineral bioleach ing heaps (SS-1 and SS-2) and 2 liquid samples recovered from bioleaching effluents (LS-1 and LS-2) were analyzed and total DNA was extracted from each sample. For all solid samples a further step was necessary, a re-puri fication of DNA, which consisted in a sample re-purification using any existing purification technique; in our laboratories this step is performed using commercial DNA purification columns to obtain a translucent appearance in the extraction Solution. Total DNA was quantified in each sample using a NanoDrop 1.0 spectrophotometer. Total extracted DNA nanograms (T) are shown in Table 1-1 together with the initial sample vol umes (C). Registered results were:
TABLE 1-1
Sample T Cn
SS-1 543.2 100 g SS-2 1660.4 20 g LS-1 11365.2 45 mL. LS-2 16364.6 45 mL.
Each of these samples was diluted with sterile nuclease-free water in order to obtain a concentration in the range 0.5 to 30 ng/ul. Table 1-2 shows the final volume to which the DNA Solution was brought and its final concentration.
TABLE 1-2
Sample Final volume (l) Concentration (ngful)
SS-1 70 7.76 SS-2 70 23.72 LS-1 421 27 LS-2 545.5 30
A calibration curve was simultaneously prepared for each taxon to allow the calculation of molecule number of the target gene in experimental samples. The standard DNA was prepared for each taxon using PCR products obtained with specific primers shown in table 1-3.
TABLE 1-3
Alignment temperature Used primers
Amplify lengths (bp)
68 C. (P.1) 533-F: 5' - GTG CCA 374
US 2011/O 1361.25 A1
TABLE 1-3 - continued
Microorganism to be Alignment determined temperature Used primers
(P. 2) 907-R: 5' - CCG TCA ATT CCT TTG. AGT T-3'
A. ferrooxidans 6 Oc C. (P.13) F : 5'- CTA GAG TAT GGG AGA. GGG TG-3
(P. 6) R: 5'- CTC TGC AGA ATT CCG GAC AT-3'
A. thiooxidans 6 o C. (P. 7) F: is "-TAA TAT CCCC TGC TGT TGA C-3'
(P.11) R: 5'-TTT CAC GAC AGA CCT AAT G-3
Leptospirillum sp. 588 C. (P. 4) F : 5'-GGA ACC GTG AAG GGT TTC G-3
(P. 2) R: 5'- CCG TCA TCG GGG ATA TTT A-3
Acidiphilium sp. 618 C. (P.10) F : 5' - GTC GCC TAA GGA. GGA, GCC T-3'
(P. 3) R: 5'-GGA GCT TAT
The PCR reaction to obtain the PCR products was carried out using as substrate the respective genomic DNA (200 ng) from each of the following microorganisms: Acidithiobacillus thiooxidans, Acidithiobacillus ferrooxidans, Leptospirillum sp. and Acidiphilium sp. The composition of PCR mix is detailed in table 1-4. The PCR product was purified using commercial DNA purification columns and concentration of the respective PCR products was determinate using Nano Drop 1.0 spectrophotometer.
TABLE 1-4
Reagent 1 reaction
Sterile nuclease-free H2O 18.35 ul PCR Buffer 10x 2.5 ul MgCl2 (50 mM) 1.5 ul dNTPs (10 mM each) 0.5 ul Primer Forward* (10 M) 0.5 ul Primer Reverse (10M) 0.5 ul Hot Start Taq (5 U?ul) 0.15 ul
*Used primers are described in Table 1-3.
More specifically, the PCR products were obtained using genomic DNA from the following strains:
(0096 A. ferrooxidans DSM 16786; 0097 A. thiooxidans DSM 504; (0098. Leptospirillum sp. DSM 1931 and (0099. Acidiphilium acidophilus DSMZ 700.
The number of molecules for each PCR product was calcu lated using the equation (2) described above. Each standard curves was prepared using five serial dilutions containing 1x10 molecules of specific PCR products in a
Jun. 9, 2011
Amplify lengths (bp)
365
146
315
263
final volume of 20 ul, which is included in the calibration CUV.
0100. A reaction “master mix” for the quantitative PCR was prepared wherein the amount of each constituent was multiplied by the total number of reactions to be carried out in order to homogenize reagent concentrations in the different PCR tubes. The reaction mix was aliquoted in 0.2 ml tubes, using a Volume of 24 Jul of reaction mix per tube. Subsequently 5 quantitative PCR were performed, one for each taxon: Acidithiobacillus thiooxidans, Acidithiobacillus ferrooxidans, Leptospirillum sp., Acidiphilium sp. and one for total bacteria, using specific primers for each of them. Sense and antisense primers were selected for the different taxons from those designed specifically by our method. Primers used for each taxon and their respective annealing temperatures are indicated in Table 1-3.
In the present Example, the following reactions were per formed in duplicate:
01.01 0102 d) Five reactions for each point of calibration curve (n-6), corresponding to standard DNA mastermix concentrations of 1x10, 1x10", 1x10, 1x10 and 1x10" molecules, and a blank.
0103) One quantitative PCR reaction was carried out on each sample and on standard DNA, these reactions being performed in duplicate. The qPCR was carried out using Mix SYBR Green qPCR. Each quantitative PCR reaction was carried out by duplicate which gives a total of 40 reactions per each taxon.
c) Five reaction for each sample (n=4) and
The quantitative PCR reaction mix for each taxon is shown in Table 1-5, where primers used are those described on Table 1-3.
US 2011/O 1361.25 A1
TABLE 1-5
1 reaction 20 reactions
Sterile nuclease-free H2O 16.1 ul 322 Jul Primer 1 (10 M) 0.5 ul 10 ul Primer 2 (10 M) 0.5 ul 10 ul PCR Buffer 10x 2.5 ul 50 ul MgCl2 (50 mM) 1.5 ul 30 ul dNTPs (10 mM each) 2.5 ul 50 ul Hot Start Taq (5 U?ul) 0.15 ul 3 ul SYBR Green qPCR 100x 0.25 ul 5 ul
This reaction mix was homogenized and aliquoted in 200.2 ml tubes, which were duly labeled. To each of the tubes 1 ul of DNA sample dilution or 1 ul of sterile nuclease-free water for the blank was added. PCR tubes containing the reaction mix and sample were shaked at Vortex for 5 seconds and centrifuged for 1 minute at 2000xg, in order to homogenize and bring the reaction liquid to the bottom of the tube, respectively. Then, the tubes with quantitative PCR reactions were sub jected to temperature cycles for amplification (table 1-6). According to the microorganism to be determined, different primer pairs were used and therefore different amplification programs were used. In the following Table, amplification programs used in the different PCR reactions are shown.
TABLE 1-6
Step Temperature (C.) Time (s)
1 Initial denaturation 95 120 2 Denaturation 95 30 3 Alignment (*) 30 4 Extension 72 40 5 Pre-reading 8O 10 6 Reading 8O
Repeat 40 times from step 2 to step 6 (qPCR cycle) 7 Denaturation curve Range between 70 and
100° C., reading each 0.2°C.
() specific temperature for each used primer pair, as indicated in Table 1-3,
When the quantitative PCR is finished, all data generated by the qPCR thermocycler are stored, from standard curve is calculated the number of molecules for target gene into each sample; this data corresponds to Q and is shown in Table 1-7. wherein DNA amounts in nanograms used for each reaction are included (U).
22 Jun. 9, 2011
0104 Calculation of the Number of Microorganisms Present in the Samples. 0105 Taking into account the qPCR result and data gen erated during the process, the following formula described as equation (3) can be applied:
QN molecules *Tng Ncult = H. "' Coleculesce: Ungi. Cn(nl)-(g)
0106 N=Estimated Concentration of microorganisms. 0107 Q-Number of Molecules as calculated with the standard curve using the C(T) value given by the equip ment.
0.108 T-Total DNA isolated from original sample. 0109 U=Amount of DNA used in the qPCR reaction. 0110 C=Number of molecules or copies by genome of the specific DNA fragment detected. (see table 1-8)
0.111 Cm=Amount in ''g'' or “ml of the processed sample
TABLE 1-8
Number of copies of 16S rDNA gene in the genome of different taxons as described in literature or determined by Southern blot.
Microorganisms C
A. ferrooxidans A. thiooxidans Acidiphillium Leptospirillaim Bacteria total
*The number of 16S rDNA copies by genome of total bacteria was estimated as an average according to our observations,
According to the previous, the following microbiological populations were determined in the analyzed samples:
TABLE 1-9
SS-1
Bacteria Mo.g. of sample
Total bacteria S.27E--OS A. ferrooxidans 1.OOE--O3 A. thiooxidans 1.29E-04 Leptospirilium sp. S.2SE--O2 Acidiphilium sp. 4.94E--04
TABLE 1-7
Q
Total Leptospirillum Acidiphilium
Sample bacteria A. ferrooxidans A. thiooxidans sp. Sp. U
SS-1 4.85E-05 O.OOSE-OS O.06E--OS O.OOSE-OS O4SE--OS 2.5
SS-2 3.48E--OS O.O47E--OS O.O13E--OS O.71E--OS O.34E--OS 2.5
LS-1 3.12E--OS O.SSE--OS O O.OO6E--OS O 2.5
LS-2 2.88E--OS O.82E--OS O O.OO6E--OS O 2.5
US 2011/O 1361.25 A1
TABLE 1-10
SS-2
Bacteria Mo.g. of sample
Total bacteria 5.78E--06
A. ferrooxidans 1.SSE--OS A. thiooxidans 4.15E-04
Leptospirilium sp. 1.17E--O6 Acidiphilium sp. S.71E--OS
TABLE 1-11
LS-1
Bacteria Mo./ml of sample
Total bacteria 1.57E--O7 A. ferrooxidans 5.61E--O6 A. thiooxidans O.OOE--OO Leptospirilium sp. 3.02E--04 Acidiphilium sp. O.OOE--OO
TABLE 1-12
LS-2
Bacteria Mo./ml of sample
Total bacteria 2.09E-07 A. ferrooxidans 1.19E--O7 A. thiooxidans O.OOE--OO Leptospirilium sp. 4.77E--04 Acidiphilium sp. O.OOE--OO
Microorganism to be determined
Total bacteria
Sulfobacillus sp.
Total archaea
Sulfolobus sp.
23
temperature Used primers
Jun. 9, 2011
Example 2 Quantification of Sulfobacillus sp., Sulfolobus sp.,
and Ferroplasma sp. in a Sample 0112 One sample obtained from mineral bioleaching heap (SS-3) and one liquid sample recovered from bioleach ing effluents (LS-3) were analyzed and total DNA was extracted from each one. A further DNA re-purification step was required to obtain a translucent appearance in the extraction solution. Total DNA was quantified in each sample using a NanoDrop 1.0 spectrophotometer. Total extracted DNA nanograms (T) are shown in Table 2-1 together with the initial sample vol umes (C).
TABLE 2-1
Sample T Cn
SS-3 537.25 20 g LS-3 1621.13 50 mL.
Each of these samples was diluted with sterile nuclease-free water in order to obtain a concentration in the range of 0.5 to 30 ng/ul. Table 2-2 shows the final volume to which the DNA Solution was brought and its final concentration.
TABLE 2-2
Sample Final volume (l) Concentration (ngful)
SS-3 70 7.67 LS-3 540 30
Five calibration curves were prepared for quantitative PCR that includes total Bacteria, Sulfobacillus, total Archaea, Fer roplasma and Sulfolobus. These calibration curves were used for calculating the molecule number of target gene in experi mental samples. The standard DNA was prepared for each taxon using PCR products obtained with specific primers described in table 2-3.
TABLE 2-3
Alignment Amplify lengths (bp)
59 (P. 1) 27-F: 5'-AGA GTT TGA 374 TCC TGG CTC AG-3'
(P. 2) 338-R 5 " - GCT GCC TCC CGT AGG AGT-3'
66 (P.1) F : 5'- CGA AGG CGG TGC 122 ACT GGC C-3
(P. 3) R: 5'-GGT GGA CCC CCG CGA CAC C-3
6 O (P.1) 515-F: 5' - GTG CCA GCA 443 GCC GCG GTA A-3
(P. 2) 958-R: 5'-TCC GGC GTT GAA TCC AAT T-3'
6 O (P. 3) F : 5' - GTC CTG GAA CGG 282 TTC CTC G-3
(P.9) R. 5" - CCC CCG CCT TTG TAG AGC G-3
US 2011/O 1361.25 A1 Jun. 9, 2011 24
TABLE 2-3 - continued
Microorganism to be Alignment Amplify determined temperature Used primers lengths (bp)
Ferroplasma sp. 56 (P.17) F : 5'-TGG CCA AGA CTT 2O4. TTC TCA T-3
(P.19) R: CTT GCA GT-3'
The PCR reaction to obtain the PCR products was carried out using the respective primers and as Substrate the respective genomic DNA (200 ng) from each of the following microor ganisms: Sulfobacillus, Ferroplasma y Sulfolobus. Substrate for PCR products for standard representing Total Archaea was obtained from the mix of the genomic DNA containing 100 ng of Ferroplasma and 100 ng of Sulfolobus. Substrate for PCR product representing Total Bacteria was obtained using DNA from Sulfobacillus (200 ng). The composition of PCR mix is detailed in table 2-4 and PCR amplification pro gram in table 2-5. The respective PCR products were purified using commercial DNA purification columns and concentra tion of them was determinate using NanoDrop 1.0 spectro photometer.
TABLE 2-4
Reagent 1 reaction
Sterile nuclease-free H2O 18.35 ul PCR Buffer 10x 2.5 ul MgCl2 (50 mM) 1.5 ul dNTPs (10 mM each) 0.5 ul Primer Forward* (10 M) 0.5 ul Primer Reverse (10M) 0.5 ul Hot Start Taq (5 U?ul) 0.15 ul
*Used primers are described in Table 2-3.
TABLE 2-5
Temperature Step (° C.) Time (s)
1. Initial denaturation 95 120 2. Denaturation 95 30 3. Alignment 30 4. Extension 72 120
Wherein steps 2 to 4 are to be repeated 28 times, The alignment temperature used is according to pairs of primer detailed in table 2-3,
The number of molecules for each PCR product was calcu lated using the equation (2) described above. Each standard curves was prepared using five serial dilutions that containing 1x10 molecules of specific PCR products in a final volume of 20 ul, which in its turn is included in the calibration curve.
0113. A reaction “master mix' for the quantitative PCR was prepared wherein the amount of each constituent was multiplied by the total number of reactions to be carried out in order to homogenize reagent concentrations in the different PCR tubes. The reaction mix was aliquoted in 0.2 ml tubes, using a Volume of 24 Jul of reaction mix per tube.
s' - CCG ATC. TCA TGT
Subsequently 5 quantitative PCR were performed, one for each taxon: Sulfobacillus, Ferroplasma, Sulfolobus, Total Bacteria and one for Total Archaea, using specific primers for each of them. Sense and antisense primers were selected for the different genera from those included in the description of the tables corresponding to each taxon. Primers used for each taxon and their respective annealing temperatures are indi cated in Table 2-3.
In the present Example, the following reactions were per formed in duplicate:
0114 0115 d) Five reactions for each point of calibration curve (n-6), corresponding to standard DNA mastermix concentrations of 1x10, 1x107, 1x10, 1x10 and 1x10" molecules, and a blank.
One quantitative PCR reaction was carried out on each sample and on standard DNA, these reactions being per formed in duplicate. The qPCR was carried out using Mix SYBR Green qPCR. Each quantitative PCR reaction was carried out by duplicate which gives a total of 16 reactions per each taxon.
c) Five reaction for each sample (n=2) and
The quantitative PCR reaction mix for each taxon is shown in Table 2-6, where primers are those that are corresponding according to Table 2-3.
TABLE 2-6
1 reaction 16 reactions
Sterile nuclease-free H2O 16.1 ul 257.6 ul Primer 1 (10 M) 0.5 ul 8 Jul Primer 2 (10 M) 0.5 ul 8 Jul PCR Buffer 10x 2.5 ul 40 ul MgCl2 (50 mM) 1.5 ul 24 ul dNTPs (10 mM each) 2.5 ul 40 ul Hot Start Taq (5 U?ul) 0.15 ul 2.4 ul SYBR Green qPCR 100x 0.25 ul 4 Il
This reaction mix was homogenized and aliquoted in 200.2 ml tubes, which were duly labeled. To each of the tubes 1 ul of DNA sample dilution or 1 ul of sterile nuclease-free water for the blank was added.
PCR tubes containing the reaction mix and sample were shaked at Vortex for 5 seconds and centrifuged for 1 minute at 2000xg, in order to homogenize and bring the reaction liquid to the bottom of the tube, respectively. Then, the tubes with quantitative PCR reactions were sub jected to temperature cycles for amplification (table 2-7).
US 2011/O 1361.25 A1 25
According to the microorganism to be determined, different primer pairs were used and therefore different amplification programs were used. In the following Table, amplification programs used in the different PCR reactions are shown.
TABLE 2-7
Step Temperature (C.) Time (s)
1 Initial denaturation 95 120 2 Denaturation 95 30 3 Alignment (*) 30 4 Extension 72 40 5 Pre-reading 8O 10 6 Reading 8O
Repeat 40 times from step 2 to step 6 (qPCR cycle) 7 Denaturation curve Between 70 and
100° C., reading each 0.2°C.
() specific temperature for each used primer pair, as indicated in Table 2-3.
When the quantitative PCR is finished, all data generated by the qPCR thermocycler are stored, from standard curve is calculated the number of molecules for target gene into each sample; this data corresponds to Q and is shown in Table 2-8. wherein DNA amounts in nanograms used for each reaction are included (U).
TABLE 2-8
Jun. 9, 2011
TABLE 2-9-continued
Number of copies of 16S rDNA gene in the genome of different taxas
Microorganisms C
Archaea total 2
Feroplasma Sulfolobus sp. 1
*The number of 16S rDNA of bacteria total and archaea total was estimated as average according our observations,
According to this, the following microbiological populations were determined in the analyzed samples:
TABLE 2-10
SS-3
Microorganism Mo.g. of sample
Total bacteria 3.09E-06 Sulfobacillus sp. 7.72E--O4
-O-
Total Sulfolobus Ferroplasma Sample Total bacteria Sulfobacillus sp. archaea sp. sp U
SS-3 S.76E--OS O.14E--OS 1.61E--OS O O.O28E--OS 2.5 LS-3 16.4E--OS O.O18E--OS 16.8E--OS 0.27E--OS 4.7SE-OS 2
0116 Calculation of the Number of Microorganisms Present in the Sample. 0117 Taking into account the qPCR result and data gen erated during the process, the following formula described as equation (3) can be applied:
N QN molecules *Tng (3) Ceit (mL), (g) Cmolecules/cel * Ung * Cn(mL)(g)
0118 N=Estimated Concentration of microorganisms. 0119 Q-Number of Molecules as calculated with the standard curve using the C(T) value given by the equip ment.
I0120 T=Total DNA isolated from original sample. I0121 U=Amount of DNA used in the qPCR reaction. 0.122 C=Number of molecules or copies by genome of the specific DNA fragment detected. (see table 1-8)
I0123. Cm=Amount in ''g'' or “ml of the processed sample
TABLE 2-9
Number of copies of 16S rRNA gene in the genome of different taxas
Microorganisms C
Bacteria total 2 Sulfobacilius 1
TABLE 2-10-continued
SS-3
Microorganism Mo.g. of sample
Total archaea 8.6SE--OS
Stilfolobus sp. O Ferroplasma sp. 3.02E--04
TABLE 2-11
LS-3
Microorganism Mo./ml of sample
Total bacteria 1.06E--08
Sulfobacillus sp. 1.18E--OS Total archaea 109E-08
Stilfolobus sp. 3.53E--06 Ferroplasma sp. 6.16E--07
US 2011/O 1361.25 A1
SEQUENCE LISTING
<16O is NUMBER OF SEO ID NOS: 417
<210s, SEQ ID NO 1 &211s LENGTH: 2O &212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: PCR primer
<4 OOs, SEQUENCE: 1
agagtttgat cctggcticag
<210s, SEQ ID NO 2 &211s LENGTH: 19 &212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: PCR primer
<4 OOs, SEQUENCE: 2
ggittaccttgttacgacitt
<210s, SEQ ID NO 3 &211s LENGTH: 2O &212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: PCR primer 22 Os. FEATURE:
<221 > NAMEAKEY: misc feature <222s. LOCATION: (14) . . (14) 223s OTHER INFORMATION: n is c or t
<4 OOs, SEQUENCE: 3
titc.cggttga t c cngc.cgga
<210s, SEQ ID NO 4 &211s LENGTH: 19 &212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Sense primer
<4 OOs, SEQUENCE: 4
Calaccacggit cqggtcaga
<210s, SEQ ID NO 5 &211s LENGTH: 19 &212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Sense primer
<4 OOs, SEQUENCE: 5
gaccttalagt tatgcgct
<210s, SEQ ID NO 6 &211s LENGTH: 19 &212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Sense primer
26
19
19
19
Jun. 9, 2011
US 2011/O 1361.25 A1
SEQUENCE: 6
agt calaccac ggtcgggt C
SEO ID NO 7 LENGTH: 2O TYPE: DNA
ORGANISM: Artificial Sequence FEATURE:
OTHER INFORMATION: Sense primer
SEQUENCE: 7
ggtttgacct taagttgatg
SEQ ID NO 8 LENGTH 19 TYPE: DNA
ORGANISM: Artificial Sequence FEATURE:
OTHER INFORMATION: Sense primer
SEQUENCE: 8
cittaagttga tigcgctaac
SEO ID NO 9 LENGTH 19 TYPE: DNA
ORGANISM: Artificial Sequence FEATURE:
OTHER INFORMATION: Sense primer
SEQUENCE: 9
ggcagt caac Cacggit cqg
SEQ ID NO 10 LENGTH 19 TYPE: DNA
ORGANISM: Artificial Sequence FEATURE:
OTHER INFORMATION: Sense primer
SEQUENCE: 1.O
cgatgctgag CtgatcCtg
SEQ ID NO 11 LENGTH 19 TYPE: DNA
ORGANISM: Artificial Sequence FEATURE:
OTHER INFORMATION: Sense primer
SEQUENCE: 11
aagttgatgc gct aaccgc
SEQ ID NO 12 LENGTH 19 TYPE: DNA
ORGANISM: Artificial Sequence FEATURE:
OTHER INFORMATION: Sense primer
SEQUENCE: 12
aaagtc.gc.ct aaggaggag
27
- Continued
19
19
19
19
19
19
Jun. 9, 2011
US 2011/O 1361.25 A1
SEQ ID NO 13 LENGTH 19 TYPE: DNA
ORGANISM: Artificial Sequence FEATURE:
OTHER INFORMATION: Sense primer
SEQUENCE: 13
gtc.gc.ctaag gaggagcct
SEQ ID NO 14 LENGTH 19 TYPE: DNA
ORGANISM: Artificial Sequence FEATURE:
OTHER INFORMATION: Sense primer
SEQUENCE: 14
aaggaggagc ctg.cgtctg
SEO ID NO 15 LENGTH 19 TYPE: DNA
ORGANISM: Artificial Sequence FEATURE:
OTHER INFORMATION: Sense primer
SEQUENCE: 15
aggagcctgc gtctgatta
SEQ ID NO 16 LENGTH: 2O TYPE: DNA
ORGANISM: Artificial Sequence FEATURE:
OTHER INFORMATION: Sense primer
SEQUENCE: 16
aggaggcagt caaccacggit
SEO ID NO 17 LENGTH 19 TYPE: DNA
ORGANISM: Artificial Sequence FEATURE:
OTHER INFORMATION: Sense primer
SEQUENCE: 17
gcgaaagt cq cctaaggag
SEQ ID NO 18 LENGTH: 2O TYPE: DNA
ORGANISM: Artificial Sequence FEATURE:
OTHER INFORMATION: Sense primer
SEQUENCE: 18
gcctaaggag gag cctgcgt
SEQ ID NO 19 LENGTH 19 TYPE: DNA
28
- Continued
19
19
19
19
Jun. 9, 2011
US 2011/O 1361.25 A1
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Sense primer
<4 OOs, SEQUENCE: 19
gcaaggaggc agt caacca
<210s, SEQ ID NO 2 O &211s LENGTH: 19 &212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Sense primer
<4 OOs, SEQUENCE: 2O
gcaagttctgct cqggcagta
<210s, SEQ ID NO 21 &211s LENGTH: 19 &212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Sense primer
<4 OOs, SEQUENCE: 21
accc.gtagga at citat cot
<210s, SEQ ID NO 22 &211s LENGTH: 2O &212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Sense primer
<4 OOs, SEQUENCE: 22
gcacagticag gcgtgaaata
<210s, SEQ ID NO 23 &211s LENGTH: 2O &212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Sense primer
<4 OOs, SEQUENCE: 23
acacatgcaa gtc.gct cqgg
<210s, SEQ ID NO 24 &211s LENGTH: 2O &212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Antisense primer
<4 OOs, SEQUENCE: 24
t citctgaccc gaccgtggitt
<210s, SEQ ID NO 25 &211s LENGTH: 2O &212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Antisense primer
29
- Continued
19
19
19
Jun. 9, 2011
US 2011/O 1361.25 A1
<4 OOs, SEQUENCE: 25
t caacttaag gtcaaaccaa
<210s, SEQ ID NO 26 &211s LENGTH: 19 &212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Antisense primer
<4 OOs, SEQUENCE: 26
ggagct tatt Ctgcgggta
<210s, SEQ ID NO 27 &211s LENGTH: 2O &212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Antisense primer
<4 OOs, SEQUENCE: 27
gcatcaactt aaggtoaaac
<210s, SEQ ID NO 28 &211s LENGTH: 2O &212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Antisense primer
<4 OOs, SEQUENCE: 28
agcgcatcaa cittaaggit ca
<210s, SEQ ID NO 29 &211s LENGTH: 2O &212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Antisense primer
<4 OOs, SEQUENCE: 29
gttagcgcat Caacttalagg
<210s, SEQ ID NO 3 O &211s LENGTH: 19 &212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Antisense primer
<4 OOs, SEQUENCE: 30
ccgaccgtgg ttgactgcc
<210s, SEQ ID NO 31 &211s LENGTH: 2O &212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Antisense primer
<4 OOs, SEQUENCE: 31
ggat cagctic agcatcgctg
<210s, SEQ ID NO 32 &211s LENGTH: 2O &212s. TYPE: DNA
30
- Continued
19
19
Jun. 9, 2011
US 2011/O 1361.25 A1
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Antisense primer
<4 OOs, SEQUENCE: 32
t caggat cag ct cagcatcg
<210s, SEQ ID NO 33 &211s LENGTH: 19 &212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Antisense primer
<4 OOs, SEQUENCE: 33
cggittagcgc atcaactta
<210s, SEQ ID NO 34 &211s LENGTH: 2O &212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Antisense primer
<4 OOs, SEQUENCE: 34
ggct cotcct tagg.cgacitt
<210s, SEQ ID NO 35 &211s LENGTH: 2O &212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Antisense primer
<4 OOs, SEQUENCE: 35
gttgactgcc ticcittgcggit
<210s, SEQ ID NO 36 &211s LENGTH: 2O &212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Antisense primer
<4 OOs, SEQUENCE: 36
t cct cottag gcgactitt cq
<210s, SEQ ID NO 37 &211s LENGTH: 2O &212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Antisense primer
<4 OO > SEQUENCE: 37
gtggttgact gcct cottgc
<210s, SEQ ID NO 38 &211s LENGTH: 2O &212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Antisense primer
31
- Continued
19
Jun. 9, 2011
US 2011/O 1361.25 A1
<4 OOs, SEQUENCE: 38
accotggttg act gcc tic ct
<210s, SEQ ID NO 39 &211s LENGTH: 2O &212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Antisense primer
<4 OOs, SEQUENCE: 39
gCaggctic ct cct taggcga
<210s, SEQ ID NO 4 O &211s LENGTH: 2O &212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Antisense primer
<4 OOs, SEQUENCE: 4 O
gacgcaggct cct cottagg
<210s, SEQ ID NO 41 &211s LENGTH: 2O &212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Antisense primer
<4 OOs, SEQUENCE: 41
t cagacgcag gct cotcc tt
<210s, SEQ ID NO 42 &211s LENGTH: 2O &212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Antisense primer
<4 OOs, SEQUENCE: 42
tgct actgcc cagogactt
<210s, SEQ ID NO 43 &211s LENGTH: 19 &212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Antisense primer
<4 OOs, SEQUENCE: 43
tgaccc.gacc gtggttgac
<210s, SEQ ID NO 44 &211s LENGTH: 19 &212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Sense primer
<4 OOs, SEQUENCE: 44
tgaggggact gcc agcgac
32
- Continued
19
19
Jun. 9, 2011
US 2011/O 1361.25 A1
<210s, SEQ ID NO 45 &211s LENGTH: 19 &212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Sense primer
<4 OOs, SEQUENCE: 45
taaat atc cc catgacgg
<210s, SEQ ID NO 46 &211s LENGTH: 2O &212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Sense primer
<4 OOs, SEQUENCE: 46
ttgtc.cggaa ccgtgaaggg
<210s, SEQ ID NO 47 &211s LENGTH: 19 &212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Sense primer
< 4 OO > SEQUENCE: 47
ggaaccgtga agggitttcg
<210s, SEQ ID NO 48 &211s LENGTH: 19 &212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Sense primer
<4 OOs, SEQUENCE: 48
ccgaat attgtc.cggalacc
<210s, SEQ ID NO 49 &211s LENGTH: 19 &212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Sense primer
<4 OOs, SEQUENCE: 49
cgacagagtt tatcgtgg
<210s, SEQ ID NO 50 &211s LENGTH: 19 &212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Sense primer
<4 OOs, SEQUENCE: 50
aatattgtcc ggalaccgtg
<210s, SEQ ID NO 51 &211s LENGTH: 19 &212s. TYPE: DNA
33
- Continued
19
19
19
19
19
Jun. 9, 2011
US 2011/O 1361.25 A1
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Sense primer
<4 OOs, SEQUENCE: 51
tccggalaccg talagggitt
<210s, SEQ ID NO 52 &211s LENGTH: 19 &212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Sense primer
<4 OOs, SEQUENCE: 52
aaatcgggcc at Cacacag
<210s, SEQ ID NO 53 &211s LENGTH: 21 &212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Sense primer
<4 OOs, SEQUENCE: 53
caaagagact ggcagactag a
<210s, SEQ ID NO 54 &211s LENGTH: 19 &212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Sense primer
<4 OOs, SEQUENCE: 54
tcgggc catc acacaggtg
<210s, SEQ ID NO 55 &211s LENGTH: 19 &212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Sense primer
<4 OO > SEQUENCE: 55
agagactggc agacitagag
<210s, SEQ ID NO 56 &211s LENGTH: 21 &212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Sense primer
<4 OOs, SEQUENCE: 56
gggggggcaa taccgaatag a
<210s, SEQ ID NO 57 &211s LENGTH: 2O &212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Sense primer
34
- Continued
19
19
21
19
19
21
Jun. 9, 2011
US 2011/O 1361.25 A1
SEQUENCE: 57
at atcaaata aatat coccg
SEO ID NO 58 LENGTH: 2O TYPE: DNA
ORGANISM: Artificial Sequence FEATURE:
OTHER INFORMATION: Sense primer
SEQUENCE: 58
aagggatat c gaataaat at
SEO ID NO 59 LENGTH: 21 TYPE: DNA
ORGANISM: Artificial Sequence FEATURE:
OTHER INFORMATION: Sense primer
SEQUENCE: 59
Ctagaggctg. g.gagagggala g
SEQ ID NO 60 LENGTH: 21 TYPE: DNA
ORGANISM: Artificial Sequence FEATURE:
OTHER INFORMATION: Sense primer
SEQUENCE: 6 O
gacgcagcaa cqc.ca.gcagt g
SEQ ID NO 61 LENGTH 19 TYPE: DNA
ORGANISM: Artificial Sequence FEATURE:
OTHER INFORMATION: Sense primer
SEQUENCE: 61
aaataaatat coccgatga
SEQ ID NO 62 LENGTH: 21 TYPE: DNA
ORGANISM: Artificial Sequence FEATURE:
OTHER INFORMATION: Sense primer
SEQUENCE: 62
Cagtgtggga agaaggctitt C
SEQ ID NO 63 LENGTH 19 TYPE: DNA
ORGANISM: Artificial Sequence FEATURE:
OTHER INFORMATION: Sense primer
SEQUENCE: 63
aacaagg tac cc.gtct aga
35
- Continued
21
21
19
21
19
Jun. 9, 2011
US 2011/O 1361.25 A1
SEQ ID NO 64 LENGTH: 2O TYPE: DNA
ORGANISM: Artificial Sequence FEATURE:
OTHER INFORMATION: Antisense primer
SEQUENCE: 64
Ctagacgggt accttgttac
SEO ID NO 65 LENGTH 19 TYPE: DNA
ORGANISM: Artificial Sequence FEATURE:
OTHER INFORMATION: Antisense primer
SEQUENCE: 65
cc.gt catcgg ggatattta
SEQ ID NO 66 LENGTH: 2O TYPE: DNA
ORGANISM: Artificial Sequence FEATURE:
OTHER INFORMATION: Antisense primer
SEQUENCE: 66
ttcacggttc cqgacaat at
SEO ID NO 67 LENGTH 19 TYPE: DNA
ORGANISM: Artificial Sequence FEATURE:
OTHER INFORMATION: Antisense primer
SEQUENCE: 67
cggttc.cgga caat attcg
SEQ ID NO 68 LENGTH: 2O TYPE: DNA
ORGANISM: Artificial Sequence FEATURE:
OTHER INFORMATION: Antisense primer
SEQUENCE: 68
c cct tcacgg titc.cggacaa
SEO ID NO 69 LENGTH: 2O TYPE: DNA
ORGANISM: Artificial Sequence FEATURE:
OTHER INFORMATION: Antisense primer
SEQUENCE: 69
ccacgatcaa act ctdtcga
SEO ID NO 7 O LENGTH: 2O TYPE: DNA
36
- Continued
19
19
Jun. 9, 2011
US 2011/O 1361.25 A1
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Antisense primer
<4 OO > SEQUENCE: 7 O
aaac cct tca cqgttc.cgga
<210s, SEQ ID NO 71 &211s LENGTH: 2O &212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Antisense primer
<4 OOs, SEQUENCE: 71
titc.cggacaa tatt cqgitat
<210s, SEQ ID NO 72 &211s LENGTH: 2O &212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Antisense primer
<4 OOs, SEQUENCE: 72
ccgaaaccct t cacggttcc
<210s, SEQ ID NO 73 &211s LENGTH: 21 &212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Antisense primer
<4 OO > SEQUENCE: 73
tagt ctdcca gtc.t.ctittgg c
<210s, SEQ ID NO 74 &211s LENGTH: 21 &212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Antisense primer
<4 OOs, SEQUENCE: 74
gCacctgtgt gatggc.ccga t
<210s, SEQ ID NO 75 &211s LENGTH: 21 &212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Antisense primer
<4 OO > SEQUENCE: 75
ctictagt ctd c cagt ct citt t
<210s, SEQ ID NO 76 &211s LENGTH: 21 &212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Antisense primer
37
- Continued
21
21
21
Jun. 9, 2011
US 2011/O 1361.25 A1
<4 OO > SEQUENCE: 76
gcagcacctg ttgatggcc C
<210s, SEQ ID NO 77 &211s LENGTH: 2O &212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Antisense primer
<4 OO > SEQUENCE: 77
Cctgttgttgat ggc.ccgattt
<210s, SEQ ID NO 78 &211s LENGTH: 21 &212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Antisense primer
<4 OO > SEQUENCE: 78
tctatt cqgt attgcc cc cc c
<210s, SEQ ID NO 79 &211s LENGTH: 21 &212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Antisense primer
<4 OO > SEQUENCE: 79
cc cctitt.cgg titc cct actic g
<210s, SEQ ID NO 8O &211s LENGTH: 21 &212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Antisense primer
<4 OOs, SEQUENCE: 80
t ccct citc cc agcctic tagt c
<210s, SEQ ID NO 81 &211s LENGTH: 19 &212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Antisense primer
<4 OOs, SEQUENCE: 81
tcqgggat at ttatttgat
<210s, SEQ ID NO 82 &211s LENGTH: 2O &212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Antisense primer
<4 OOs, SEQUENCE: 82
Cataccttgg gcggct Coct
38
- Continued
21
21
21
21
19
Jun. 9, 2011
US 2011/O 1361.25 A1
SEQ ID NO 83 LENGTH 19 TYPE: DNA
ORGANISM: Artificial Sequence FEATURE:
OTHER INFORMATION: Antisense primer
SEQUENCE: 83
cago ct ctag totgc.cagt
SEQ ID NO 84 LENGTH 19 TYPE: DNA
ORGANISM: Artificial Sequence FEATURE:
OTHER INFORMATION: Sense primer
SEQUENCE: 84
cgaaggcggit gcactggcc
SEO ID NO 85 LENGTH 19 TYPE: DNA
ORGANISM: Artificial Sequence FEATURE:
OTHER INFORMATION: Sense primer
SEQUENCE: 85
gtgg.cgalagg C9gtgcact
SEQ ID NO 86 LENGTH: 2O TYPE: DNA
ORGANISM: Artificial Sequence FEATURE:
OTHER INFORMATION: Sense primer
SEQUENCE: 86
aggtgtc.gcg ggggtccacc
SEO ID NO 87 LENGTH 19 TYPE: DNA
ORGANISM: Artificial Sequence FEATURE:
OTHER INFORMATION: Sense primer
SEQUENCE: 87
tgtctgtcgg gacgaggac
SEO ID NO 88 LENGTH 19 TYPE: DNA
ORGANISM: Artificial Sequence FEATURE:
OTHER INFORMATION: Sense primer
SEQUENCE: 88
gagggcagga gaggtgcat
SEO ID NO 89 LENGTH 19 TYPE: DNA
39
- Continued
19
19
19
19
19
Jun. 9, 2011
US 2011/O 1361.25 A1
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Sense primer
<4 OOs, SEQUENCE: 89
<210s, SEQ ID NO 90 &211s LENGTH: 19 &212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Sense primer
<4 OOs, SEQUENCE: 90
Caccitcgcgg to cqgagc
<210s, SEQ ID NO 91 &211s LENGTH: 2O &212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Sense primer
<4 OOs, SEQUENCE: 91
gggggtCCaC Ctcgcggtgc
<210s, SEQ ID NO 92 &211s LENGTH: 19 &212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Sense primer
<4 OOs, SEQUENCE: 92
Ctcgcggtgc cqgagctaa
<210s, SEQ ID NO 93 &211s LENGTH: 19 &212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Sense primer
<4 OOs, SEQUENCE: 93
tgtc.gcgggg gtccacct c
<210s, SEQ ID NO 94 &211s LENGTH: 19 &212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Sense primer
<4 OOs, SEQUENCE: 94
ggattacgagg titcgcggg
<210s, SEQ ID NO 95 &211s LENGTH: 19 &212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Sense primer
40
- Continued
19
19
19
19
19
Jun. 9, 2011
US 2011/O 1361.25 A1
SEQUENCE: 95
cggagctaac gcacticagt
SEO ID NO 96 LENGTH: 2O TYPE: DNA
ORGANISM: Artificial Sequence FEATURE:
OTHER INFORMATION: Sense primer
SEQUENCE: 96
gtaaacgatg gatacgaggit
SEO ID NO 97 LENGTH 19 TYPE: DNA
ORGANISM: Artificial Sequence FEATURE:
OTHER INFORMATION: Sense primer
SEQUENCE: 97
tgagtggggg at atcgggc
SEO ID NO 98 LENGTH 19 TYPE: DNA
ORGANISM: Artificial Sequence FEATURE:
OTHER INFORMATION: Sense primer
SEQUENCE: 98
SEO ID NO 99 LENGTH 19 TYPE: DNA
ORGANISM: Artificial Sequence FEATURE:
OTHER INFORMATION: Sense primer
SEQUENCE: 99
agctaacgca ct cagtatic
SEQ ID NO 100 LENGTH: 2O TYPE: DNA
ORGANISM: Artificial Sequence FEATURE:
OTHER INFORMATION: Sense primer
SEQUENCE: 1.OO
acgatggata caggtgtcg
SEQ ID NO 101 LENGTH: 2O TYPE: DNA
ORGANISM: Artificial Sequence FEATURE:
OTHER INFORMATION: Sense primer
SEQUENCE: 101
gtgc.cggagc taacgcactic
41
- Continued
19
19
19
19
Jun. 9, 2011
US 2011/O 1361.25 A1
SEQ ID NO 102 LENGTH: 2O TYPE: DNA
ORGANISM: Artificial Sequence FEATURE:
OTHER INFORMATION: Sense primer
SEQUENCE: 1 O2
aggtgcatgg aatticctggit
SEQ ID NO 103 LENGTH: 2O TYPE: DNA
ORGANISM: Artificial Sequence FEATURE:
OTHER INFORMATION: Sense primer
SEQUENCE: 103
tgcatggaat tcc tigtgga
SEQ ID NO 104 LENGTH 19 TYPE: DNA
ORGANISM: Artificial Sequence FEATURE:
OTHER INFORMATION: Antisense primer
SEQUENCE: 104
cagtgcaccg cct tcgc.ca
SEO ID NO 105 LENGTH 19 TYPE: DNA
ORGANISM: Artificial Sequence FEATURE:
OTHER INFORMATION: Antisense primer
SEQUENCE: 105
ggc.cagtgca ccgc.ct tcg
SEQ ID NO 106 LENGTH 19 TYPE: DNA
ORGANISM: Artificial Sequence FEATURE:
OTHER INFORMATION: Antisense primer
SEQUENCE: 106
ggtggaccCC cqcgacacc
SEO ID NO 107 LENGTH 19 TYPE: DNA
ORGANISM: Artificial Sequence FEATURE:
OTHER INFORMATION: Antisense primer
SEQUENCE: 107
ggtc.ct citc cc.gacagac
SEQ ID NO 108 LENGTH: 2O TYPE: DNA
42
- Continued
19
19
19
19
Jun. 9, 2011
US 2011/O 1361.25 A1
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Antisense primer
<4 OOs, SEQUENCE: 108
catgcacctic ticctg.ccct c
<210s, SEQ ID NO 109 &211s LENGTH: 2O &212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Antisense primer
<4 OOs, SEQUENCE: 109
ttagct Cogg Caccgc.gagg
<210s, SEQ ID NO 110 &211s LENGTH: 19 &212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Antisense primer
<4 OOs, SEQUENCE: 110
gcgaggtgga CCC cc.gcga
<210s, SEQ ID NO 111 &211s LENGTH: 19 &212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Antisense primer
<4 OOs, SEQUENCE: 111
tgcaccgcct tcgccaccg
<210s, SEQ ID NO 112 &211s LENGTH: 2O &212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Antisense primer
<4 OOs, SEQUENCE: 112
cgitatic catc gtttacggcg
<210s, SEQ ID NO 113 &211s LENGTH: 2O &212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Antisense primer
<4 OOs, SEQUENCE: 113
gaccc.ccg.cg acacct cqta
<210s, SEQ ID NO 114 &211s LENGTH: 2O &212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Antisense primer
43
- Continued
19
19
Jun. 9, 2011
US 2011/O 1361.25 A1
SEQUENCE: 114
gagtgcgitta gct Coggcac
SEQ ID NO 115 LENGTH 19 TYPE: DNA
ORGANISM: Artificial Sequence FEATURE:
OTHER INFORMATION: Antisense primer
SEQUENCE: 115
tccaccagga attic catgc
SEQ ID NO 116 LENGTH 19 TYPE: DNA
ORGANISM: Artificial Sequence FEATURE:
OTHER INFORMATION: Antisense primer
SEQUENCE: 116
gcc aggc.cag to accgc.c
SEO ID NO 117 LENGTH: 2O TYPE: DNA
ORGANISM: Artificial Sequence FEATURE:
OTHER INFORMATION: Antisense primer
SEQUENCE: 117
c caggaattic catgcacctic
SEQ ID NO 118 LENGTH: 2O TYPE: DNA
ORGANISM: Artificial Sequence FEATURE:
OTHER INFORMATION: Antisense primer
SEQUENCE: 118
cct cqtat co atcgtttacg
SEQ ID NO 119 LENGTH: 2O TYPE: DNA
ORGANISM: Artificial Sequence FEATURE:
OTHER INFORMATION: Antisense primer
SEQUENCE: 119
actgagtgcg ttagct Cogg
SEQ ID NO 120 LENGTH: 2O TYPE: DNA
ORGANISM: Artificial Sequence FEATURE:
OTHER INFORMATION: Antisense primer
SEQUENCE: 12O
gatactgagt gcgittagctic
44
- Continued
19
19
Jun. 9, 2011
US 2011/O 1361.25 A1
<210s, SEQ ID NO 121 &211s LENGTH: 2O &212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Antisense primer
<4 OOs, SEQUENCE: 121
gccacacctic gitat coat cq
<210s, SEQ ID NO 122 &211s LENGTH: 2O &212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Antisense primer
<4 OOs, SEQUENCE: 122
cgggat actg agtgcgittag
<210s, SEQ ID NO 123 &211s LENGTH: 19 &212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Antisense primer
< 4 OO > SEQUENCE: 123
gcc.cgatat c ccc cactica
<210s, SEQ ID NO 124 &211s LENGTH: 19 &212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Sense primer
<4 OOs, SEQUENCE: 124
cagtgcaccg cct tcgc.ca
<210s, SEQ ID NO 125 &211s LENGTH: 19 &212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Sense primer
<4 OOs, SEQUENCE: 125
ggc.cagtgca ccgc.ct tcg
<210s, SEQ ID NO 126 &211s LENGTH: 19 &212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Sense primer
<4 OOs, SEQUENCE: 126
ggtggaccCC cqcgacacc
<210s, SEQ ID NO 127 &211s LENGTH: 19 &212s. TYPE: DNA
45
- Continued
19
19
19
19
Jun. 9, 2011
US 2011/O 1361.25 A1
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Sense primer
<4 OOs, SEQUENCE: 127
ggtc.ct citc cc.gacagac
<210s, SEQ ID NO 128 &211s LENGTH: 2O &212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Sense primer
<4 OOs, SEQUENCE: 128
catgcacctic ticctg.ccct c
<210s, SEQ ID NO 129 &211s LENGTH: 2O &212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Sense primer
<4 OOs, SEQUENCE: 129
ttagct Cogg Caccgc.gagg
<210s, SEQ ID NO 130 &211s LENGTH: 19 &212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Sense primer
<4 OOs, SEQUENCE: 130
gcgaggtgga CCC cc.gcga
<210s, SEQ ID NO 131 &211s LENGTH: 19 &212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Sense primer
<4 OOs, SEQUENCE: 131
tgcaccgcct tcgccaccg
<210s, SEQ ID NO 132 &211s LENGTH: 2O &212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Sense primer
<4 OOs, SEQUENCE: 132
cgitatic catc gtttacggcg
<210s, SEQ ID NO 133 &211s LENGTH: 2O &212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Sense primer
46
- Continued
19
19
19
Jun. 9, 2011
US 2011/O 1361.25 A1
<4 OOs, SEQUENCE: 133
gaccc.ccg.cg acacct cqta
<210s, SEQ ID NO 134 &211s LENGTH: 2O &212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Sense primer
<4 OOs, SEQUENCE: 134
gagtgcgitta gct Coggcac
<210s, SEQ ID NO 135 &211s LENGTH: 19 &212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Sense primer
<4 OOs, SEQUENCE: 135
tccaccagga attic catgc
<210s, SEQ ID NO 136 &211s LENGTH: 19 &212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Sense primer
<4 OOs, SEQUENCE: 136
gcc aggc.cag to accgc.c
<210s, SEQ ID NO 137 &211s LENGTH: 2O &212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Sense primer
<4 OOs, SEQUENCE: 137
c caggaattic catgcacctic
<210s, SEQ ID NO 138 &211s LENGTH: 2O &212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Sense primer
<4 OOs, SEQUENCE: 138
cct cqtat co atcgtttacg
<210s, SEQ ID NO 139 &211s LENGTH: 2O &212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Sense primer
<4 OOs, SEQUENCE: 139
actgagtgcg ttagct Cogg
47
- Continued
19
19
Jun. 9, 2011
US 2011/O 1361.25 A1
<210s, SEQ ID NO 140 &211s LENGTH: 2O &212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Sense primer
<4 OOs, SEQUENCE: 140
gatactgagt gcgittagctic
<210s, SEQ ID NO 141 &211s LENGTH: 2O &212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Sense primer
<4 OOs, SEQUENCE: 141
gccacacctic gitat coat cq
<210s, SEQ ID NO 142 &211s LENGTH: 2O &212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Sense primer
< 4 OO > SEQUENCE: 142
cgggat actg agtgcgittag
<210s, SEQ ID NO 143 &211s LENGTH: 19 &212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Sense primer
<4 OOs, SEQUENCE: 143
gcc.cgatat c ccc cactica
<210s, SEQ ID NO 144 &211s LENGTH: 19 &212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Antisense primer
<4 OOs, SEQUENCE: 144
cagtgcaccg cct tcgc.ca
<210s, SEQ ID NO 145 &211s LENGTH: 19 &212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Antisense primer
<4 OOs, SEQUENCE: 145
ggc.cagtgca ccgc.ct tcg
<210s, SEQ ID NO 146 &211s LENGTH: 19 &212s. TYPE: DNA
48
- Continued
19
19
19
Jun. 9, 2011
US 2011/O 1361.25 A1
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Antisense primer
<4 OOs, SEQUENCE: 146
ggtggaccCC cqcgacacc
<210s, SEQ ID NO 147 &211s LENGTH: 19 &212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Antisense primer
<4 OOs, SEQUENCE: 147
ggtc.ct citc cc.gacagac
<210s, SEQ ID NO 148 &211s LENGTH: 2O &212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Antisense primer
<4 OOs, SEQUENCE: 148
catgcacctic ticctg.ccct c
<210s, SEQ ID NO 149 &211s LENGTH: 2O &212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Antisense primer
<4 OOs, SEQUENCE: 149
ttagct Cogg Caccgc.gagg
<210s, SEQ ID NO 150 &211s LENGTH: 19 &212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Antisense primer
<4 OOs, SEQUENCE: 150
gcgaggtgga CCC cc.gcga
<210s, SEQ ID NO 151 &211s LENGTH: 19 &212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Antisense primer
<4 OOs, SEQUENCE: 151
tgcaccgcct tcgccaccg
<210s, SEQ ID NO 152 &211s LENGTH: 2O &212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Antisense primer
49
- Continued
19
19
19
19
Jun. 9, 2011
US 2011/O 1361.25 A1
<4 OOs, SEQUENCE: 152
cgitatic catc gtttacggcg
<210s, SEQ ID NO 153 &211s LENGTH: 2O &212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Antisense primer
<4 OOs, SEQUENCE: 153
gaccc.ccg.cg acacct cqta
<210s, SEQ ID NO 154 &211s LENGTH: 2O &212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Antisense primer
<4 OOs, SEQUENCE: 154
gagtgcgitta gct Coggcac
<210s, SEQ ID NO 155 &211s LENGTH: 19 &212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Antisense primer
<4 OO > SEQUENCE: 155
tccaccagga attic catgc
<210s, SEQ ID NO 156 &211s LENGTH: 19 &212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Antisense primer
<4 OOs, SEQUENCE: 156
gcc aggc.cag to accgc.c
<210s, SEQ ID NO 157 &211s LENGTH: 2O &212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Antisense primer
<4 OO > SEQUENCE: 157
c caggaattic catgcacctic
<210s, SEQ ID NO 158 &211s LENGTH: 2O &212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Antisense primer
<4 OOs, SEQUENCE: 158
cct cqtat co atcgtttacg
50
- Continued
19
19
Jun. 9, 2011
US 2011/O 1361.25 A1
<210s, SEQ ID NO 159 &211s LENGTH: 2O &212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Antisense primer
<4 OOs, SEQUENCE: 159
actgagtgcg ttagct Cogg
<210s, SEQ ID NO 160 &211s LENGTH: 2O &212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Antisense primer
<4 OOs, SEQUENCE: 160
gatactgagt gcgittagctic
<210s, SEQ ID NO 161 &211s LENGTH: 2O &212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Antisense primer
< 4 OO > SEQUENCE: 161
gccacacctic gitat coat cq
<210s, SEQ ID NO 162 &211s LENGTH: 2O &212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Antisense primer
<4 OOs, SEQUENCE: 162
cgggat actg agtgcgittag
<210s, SEQ ID NO 163 &211s LENGTH: 19 &212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Antisense primer
<4 OOs, SEQUENCE: 163
gcc.cgatat c ccc cactica
<210s, SEQ ID NO 164 &211s LENGTH: 2O &212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Sense primer
<4 OOs, SEQUENCE: 164
gggagacgaa alaggtaatcg
<210s, SEQ ID NO 165 &211s LENGTH: 2O &212s. TYPE: DNA
51
- Continued
19
Jun. 9, 2011
US 2011/O 1361.25 A1
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Sense primer
<4 OOs, SEQUENCE: 165
aaagttctitt C9gtgacggg
<210s, SEQ ID NO 166 &211s LENGTH: 2O &212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Sense primer
<4 OOs, SEQUENCE: 166
cggggalaggt tatatgtta
<210s, SEQ ID NO 167 &211s LENGTH: 19 &212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Sense primer
<4 OOs, SEQUENCE: 167
gagggagalaa CC9ggggat
<210s, SEQ ID NO 168 &211s LENGTH: 19 &212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Sense primer
<4 OOs, SEQUENCE: 168
aatcgctaat atcggittac
<210s, SEQ ID NO 169 &211s LENGTH: 2O &212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Sense primer
<4 OOs, SEQUENCE: 169
ccgggggatc titcggacctic
<210s, SEQ ID NO 170 &211s LENGTH: 2O &212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Sense primer
<4 OOs, SEQUENCE: 170
taatat cqcc togctgttgac
<210s, SEQ ID NO 171 &211s LENGTH: 19 &212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Sense primer
52
- Continued
19
19
Jun. 9, 2011
US 2011/O 1361.25 A1
<4 OOs, SEQUENCE: 171
tCggtgacgg galaggttg
<210s, SEQ ID NO 172 &211s LENGTH: 19 &212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Sense primer
<4 OOs, SEQUENCE: 172
ggagaalaccg ggggat Ctt
<210s, SEQ ID NO 173 &211s LENGTH: 2O &212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Sense primer
<4 OOs, SEQUENCE: 173
acgt.cctgag ggagaalaccg
<210s, SEQ ID NO 174 &211s LENGTH: 2O &212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Sense primer
<4 OOs, SEQUENCE: 174
agacgaaaag gtaatcgcta
<210s, SEQ ID NO 175 &211s LENGTH: 19 &212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Sense primer
<4 OO > SEQUENCE: 175
gtgacgggga aggttgata
<210s, SEQ ID NO 176 &211s LENGTH: 19 &212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Sense primer
<4 OOs, SEQUENCE: 176
gaaaccgggg gat ctitcgg
<210s, SEQ ID NO 177 &211s LENGTH: 2O &212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Sense primer
<4 OO > SEQUENCE: 177
tCctgaggga gaalaccgggg
53
- Continued
19
19
19
19
Jun. 9, 2011
US 2011/O 1361.25 A1
SEO ID NO 178 LENGTH: 2O TYPE: DNA
ORGANISM: Artificial Sequence FEATURE:
OTHER INFORMATION: Sense primer
SEQUENCE: 178
cgaaaaggta atcgctaata
SEO ID NO 179 LENGTH: 2O TYPE: DNA
ORGANISM: Artificial Sequence FEATURE:
OTHER INFORMATION: Sense primer
SEQUENCE: 179
aaaggtaatc gctaatat cq
SEQ ID NO 18O LENGTH: 2O TYPE: DNA
ORGANISM: Artificial Sequence FEATURE:
OTHER INFORMATION: Sense primer
SEQUENCE: 180
tcgtgggaga caaaaggta
SEQ ID NO 181 LENGTH: 2O TYPE: DNA
ORGANISM: Artificial Sequence FEATURE:
OTHER INFORMATION: Sense primer
SEQUENCE: 181
cggacct cqt gct attggag
SEQ ID NO 182 LENGTH 19 TYPE: DNA
ORGANISM: Artificial Sequence FEATURE:
OTHER INFORMATION: Sense primer
SEQUENCE: 182
gttctttcgg tacgggga
SEQ ID NO 183 LENGTH 19 TYPE: DNA
ORGANISM: Artificial Sequence FEATURE:
OTHER INFORMATION: Sense primer
SEQUENCE: 183
Cttt C9gtga C9gggalagg
SEQ ID NO 184 LENGTH 19 TYPE: DNA
54
- Continued
19
19
Jun. 9, 2011
US 2011/O 1361.25 A1
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Antisense primer
<4 OOs, SEQUENCE: 184
atc.ccc.cggit ttcticcict c
<210s, SEQ ID NO 185 &211s LENGTH: 19 &212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Antisense primer
<4 OOs, SEQUENCE: 185
at attagciga ttacct titt
<210s, SEQ ID NO 186 &211s LENGTH: 2O &212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Antisense primer
<4 OOs, SEQUENCE: 186
caacct tccc cqt caccgaa
<210s, SEQ ID NO 187 &211s LENGTH: 2O &212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Antisense primer
<4 OOs, SEQUENCE: 187
ccgaagat.co. cccggtttct
<210s, SEQ ID NO 188 &211s LENGTH: 2O &212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Antisense primer
<4 OOs, SEQUENCE: 188
CtcCaatagc acgaggtocg
<210s, SEQ ID NO 189 &211s LENGTH: 2O &212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Antisense primer
<4 OOs, SEQUENCE: 189
accoat atta gcgattacct
<210s, SEQ ID NO 190 &211s LENGTH: 19 &212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Antisense primer
55
- Continued
19
19
Jun. 9, 2011
US 2011/O 1361.25 A1
<4 OOs, SEQUENCE: 190
aagat.ccc cc ggtttcticc
<210s, SEQ ID NO 191 &211s LENGTH: 2O &212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Antisense primer
<4 OOs, SEQUENCE: 191
tat caacct t c ccc.gt cacc
<210s, SEQ ID NO 192 &211s LENGTH: 2O &212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Antisense primer
<4 OOs, SEQUENCE: 192
ggtttctic cc ticaggacgta
<210s, SEQ ID NO 193 &211s LENGTH: 2O &212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Antisense primer
<4 OOs, SEQUENCE: 193
ggtc.cgaaga t ccc.ccggitt
<210s, SEQ ID NO 194 &211s LENGTH: 19 &212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Antisense primer
<4 OOs, SEQUENCE: 194
titt cacgaca gacctaatg
<210s, SEQ ID NO 195 &211s LENGTH: 2O &212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Antisense primer
<4 OOs, SEQUENCE: 195
gtaaccata ttagcgatta
<210s, SEQ ID NO 196 &211s LENGTH: 2O &212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Antisense primer
<4 OOs, SEQUENCE: 196
acatat caac ct tcc.ccgtc
56
- Continued
19
19
Jun. 9, 2011
US 2011/O 1361.25 A1
<210s, SEQ ID NO 197 &211s LENGTH: 2O &212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Antisense primer
<4 OO > SEQUENCE: 197
cc.cggtttct c cct caggac
<210s, SEQ ID NO 198 &211s LENGTH: 2O &212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Antisense primer
<4 OOs, SEQUENCE: 198
gcqattacct titt cqt ct co
<210s, SEQ ID NO 199 &211s LENGTH: 2O &212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Antisense primer
< 4 OO > SEQUENCE: 199
cc.ccgt cacc gaaagaactt
<210s, SEQ ID NO 2 OO &211s LENGTH: 2O &212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Antisense primer
<4 OOs, SEQUENCE: 2OO
ttalacatlatc. aac Ctt CCCC
<210s, SEQ ID NO 2 O1 &211s LENGTH: 2O &212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Antisense primer
<4 OOs, SEQUENCE: 2O1
ttagcgatta ccttitt cqtc
<210s, SEQ ID NO 202 &211s LENGTH: 2O &212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Antisense primer
<4 OOs, SEQUENCE: 2O2
citt.ccc.cgt.c accogaaagaa
<210s, SEQ ID NO 203 &211s LENGTH: 2O &212s. TYPE: DNA
57
- Continued
Jun. 9, 2011
US 2011/O 1361.25 A1
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Antisense primer
<4 OOs, SEQUENCE: 2O3
attaccttitt cqt citcc.cac
<210s, SEQ ID NO 204 &211s LENGTH: 19 &212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Sense primer
<4 OOs, SEQUENCE: 204
gggaaa.ccgt gagggcgct
<210s, SEQ ID NO 205 &211s LENGTH: 2O &212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Sense primer
<4 OOs, SEQUENCE: 205
gcgaaacgtc. cccaatgcgg
<210s, SEQ ID NO 206 &211s LENGTH: 21 &212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Sense primer
<4 OOs, SEQUENCE: 2O6
cc.gcagggaa accggtaagc C
<210s, SEQ ID NO 2 O7 &211s LENGTH: 2O &212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Sense primer
<4 OOs, SEQUENCE: 2O7
cc.cgggaaag ggcagtgata
<210s, SEQ ID NO 208 &211s LENGTH: 19 &212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Sense primer
<4 OOs, SEQUENCE: 208
gggaaagggc agtgat act
<210s, SEQ ID NO 209 &211s LENGTH: 2O &212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Sense primer
58
- Continued
19
21
19
Jun. 9, 2011
US 2011/O 1361.25 A1
<4 OOs, SEQUENCE: 209
aatc.cggggg agg.cgaaggg
<210s, SEQ ID NO 210 &211s LENGTH: 2O &212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Sense primer
<4 OOs, SEQUENCE: 210
aggg tactgg aacgt.ccctt
<210s, SEQ ID NO 211 &211s LENGTH: 21 &212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Sense primer
<4 OOs, SEQUENCE: 211
aagcgt.ccgg C cagaacg.cg C
<210s, SEQ ID NO 212 &211s LENGTH: 2O &212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Sense primer
<4 OOs, SEQUENCE: 212
cgc.ctaaagg ggcatgggct
<210s, SEQ ID NO 213 &211s LENGTH: 2O &212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Sense primer
<4 OOs, SEQUENCE: 213
ggct attt co cqct catgcc
<210s, SEQ ID NO 214 &211s LENGTH: 21 &212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Sense primer
<4 OOs, SEQUENCE: 214
cgitacgcc ct cqggtaagag g
<210s, SEQ ID NO 215 &211s LENGTH: 2O &212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Sense primer
<4 OOs, SEQUENCE: 215
aacggc.ccgc caaaccgata
59
- Continued
21
21
Jun. 9, 2011
US 2011/O 1361.25 A1
<210s, SEQ ID NO 216 &211s LENGTH: 19 &212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Sense primer
<4 OOs, SEQUENCE: 216
agc.cggcc ct gcaagt cac
<210s, SEQ ID NO 217 &211s LENGTH: 19 &212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Sense primer
<4 OOs, SEQUENCE: 217
Cactgcttaa agaccc.ggg
<210s, SEQ ID NO 218 &211s LENGTH: 21 &212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Sense primer
< 4 OO > SEQUENCE: 218
ggagctaatc C9gggCaggc g
<210s, SEQ ID NO 219 &211s LENGTH: 2O &212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Sense primer
<4 OOs, SEQUENCE: 219
aalaccgtgag ggcgct accc
<210s, SEQ ID NO 220 &211s LENGTH: 21 &212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Sense primer
<4 OOs, SEQUENCE: 220
aggcgaaggg tactggaacg t
<210s, SEQ ID NO 221 &211s LENGTH: 2O &212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Sense primer
<4 OOs, SEQUENCE: 221
acccc.cagtg Ctc.ccgaaag
<210s, SEQ ID NO 222 &211s LENGTH: 21 &212s. TYPE: DNA
60
- Continued
19
19
21
21
Jun. 9, 2011
US 2011/O 1361.25 A1
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Sense primer
<4 OOs, SEQUENCE: 222
CCCttcgc.ct aaaggggcat g
<210s, SEQ ID NO 223 &211s LENGTH: 21 &212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Sense primer
<4 OOs, SEQUENCE: 223
gcatgggcta titt cocqctic a
<210s, SEQ ID NO 224 &211s LENGTH: 19 &212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Sense primer
<4 OOs, SEQUENCE: 224
gggaaa.ccgt gagggcgct
<210s, SEQ ID NO 225 &211s LENGTH: 2O &212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Sense primer
<4 OOs, SEQUENCE: 225
gcgaaacgtc. cccaatgcgg
<210s, SEQ ID NO 226 &211s LENGTH: 21 &212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Antisense primer
<4 OOs, SEQUENCE: 226
cc.gcattggg gacgtttcgc g
<210s, SEQ ID NO 227 &211s LENGTH: 21 &212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Antisense primer
<4 OOs, SEQUENCE: 227
gcgc.cct cac ggttt ccc.gc a
<210s, SEQ ID NO 228 &211s LENGTH: 21 &212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Antisense primer
61
- Continued
21
21
19
21
21
Jun. 9, 2011
US 2011/O 1361.25 A1
SEQUENCE: 228
cc.gcattggg gacgtttcgc g
SEQ ID NO 229 LENGTH: 21 TYPE: DNA
ORGANISM: Artificial Sequence FEATURE:
OTHER INFORMATION: Antisense primer
SEQUENCE: 229
gcgc.cct cac ggttt ccc.gc a
SEQ ID NO 230 LENGTH: 21 TYPE: DNA
ORGANISM: Artificial Sequence FEATURE:
OTHER INFORMATION: Antisense primer
SEQUENCE: 23 O
titc.ccgcatt ggggacgttt C
SEQ ID NO 231 LENGTH: 2O TYPE: DNA
ORGANISM: Artificial Sequence FEATURE:
OTHER INFORMATION: Antisense primer
SEQUENCE: 231
tagcgc cct c acggtttccc
SEQ ID NO 232 LENGTH: 2O TYPE: DNA
ORGANISM: Artificial Sequence FEATURE:
OTHER INFORMATION: Antisense primer
SEQUENCE: 232
ggct taccgg ttt Coctg.cg
SEQ ID NO 233 LENGTH 19 TYPE: DNA
ORGANISM: Artificial Sequence FEATURE:
OTHER INFORMATION: Antisense primer
SEQUENCE: 233
ctg.ccc.tttic ccgggttga
SEQ ID NO 234 LENGTH 19 TYPE: DNA
ORGANISM: Artificial Sequence FEATURE:
OTHER INFORMATION: Antisense primer
SEQUENCE: 234
t cactg.ccct titc.ccgggit
62
- Continued
21
21
21
19
19
Jun. 9, 2011
US 2011/O 1361.25 A1
SEO ID NO 235 LENGTH 19 TYPE: DNA
ORGANISM: Artificial Sequence FEATURE:
OTHER INFORMATION: Antisense primer
SEQUENCE: 235
gtat cactgc cctitt.ccc.g
SEQ ID NO 236 LENGTH: 2O TYPE: DNA
ORGANISM: Artificial Sequence FEATURE:
OTHER INFORMATION: Antisense primer
SEQUENCE: 236
gcc.cgggit ct ttaa.gcagtg
SEO ID NO 237 LENGTH: 21 TYPE: DNA
ORGANISM: Artificial Sequence FEATURE:
OTHER INFORMATION: Antisense primer
SEQUENCE: 237
citcc.cgcc cc ctagocct go a
SEQ ID NO 238 LENGTH: 21 TYPE: DNA
ORGANISM: Artificial Sequence FEATURE:
OTHER INFORMATION: Antisense primer
SEQUENCE: 238
cc.cgggat.ct gtggattt cq c
SEQ ID NO 239 LENGTH 19 TYPE: DNA
ORGANISM: Artificial Sequence FEATURE:
OTHER INFORMATION: Antisense primer
SEQUENCE: 239
taccc.gaggg C9tacgact
SEQ ID NO 240 LENGTH: 21 TYPE: DNA
ORGANISM: Artificial Sequence FEATURE:
OTHER INFORMATION: Antisense primer
SEQUENCE: 24 O
Cctictt accc gagggcgtac g
SEQ ID NO 241 LENGTH 19 TYPE: DNA
63
- Continued
19
21
21
19
21
Jun. 9, 2011
US 2011/O 1361.25 A1
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Antisense primer
<4 OOs, SEQUENCE: 241
titcgc.ctgcc ccggattag
<210s, SEQ ID NO 242 &211s LENGTH: 21 &212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Antisense primer
<4 OOs, SEQUENCE: 242
ggcggCaggc titaccggittt C
<210s, SEQ ID NO 243 &211s LENGTH: 21 &212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Antisense primer
<4 OOs, SEQUENCE: 243
cggattagct c cagtttic cc g
<210s, SEQ ID NO 244 &211s LENGTH: 19 &212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Antisense primer
<4 OOs, SEQUENCE: 244
ggacgttcca gtacccttic
<210s, SEQ ID NO 245 &211s LENGTH: 2O &212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Antisense primer
<4 OOs, SEQUENCE: 245
cc.ccggatta gct coagttt
<210s, SEQ ID NO 246 &211s LENGTH: 21 &212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Antisense primer
<4 OOs, SEQUENCE: 246
tacic ctitcgc ctdcc.ccgga t
<210s, SEQ ID NO 247 &211s LENGTH: 19 &212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Antisense primer
64
- Continued
19
21
21
19
21
Jun. 9, 2011
US 2011/O 1361.25 A1
<4 OOs, SEQUENCE: 247
c catgc.ccct ttagg.cgaa
<210s, SEQ ID NO 248 &211s LENGTH: 21 &212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Sense primer
<4 OOs, SEQUENCE: 248
agagt caa.cc tacgagctt a
<210s, SEQ ID NO 249 &211s LENGTH: 21 &212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Sense primer
<4 OOs, SEQUENCE: 249
gtcaacct ga cdagct tact c
<210s, SEQ ID NO 250 &211s LENGTH: 2O &212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Sense primer
<4 OOs, SEQUENCE: 250
tgagagt caa cctgacgagc
<210s, SEQ ID NO 251 &211s LENGTH: 21 &212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Sense primer
<4 OOs, SEQUENCE: 251
gagott actic gataggagga g
<210s, SEQ ID NO 252 &211s LENGTH: 19 &212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Sense primer
<4 OOs, SEQUENCE: 252
tittaatticga gagggittaa
<210s, SEQ ID NO 253 &211s LENGTH: 21 &212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Sense primer
<4 OOs, SEQUENCE: 253
Cttact cat agcaggagag g
65
- Continued
19
21
21
21
19
21
Jun. 9, 2011
US 2011/O 1361.25 A1
SEQ ID NO 254 LENGTH: 21 TYPE: DNA
ORGANISM: Artificial Sequence FEATURE:
OTHER INFORMATION: Sense primer
SEQUENCE: 254
aatcaaatct gatgtcggtg a
SEO ID NO 255 LENGTH 19 TYPE: DNA
ORGANISM: Artificial Sequence FEATURE:
OTHER INFORMATION: Sense primer
SEQUENCE: 255
ggittaaatca aatctgatg
SEQ ID NO 256 LENGTH: 21 TYPE: DNA
ORGANISM: Artificial Sequence FEATURE:
OTHER INFORMATION: Sense primer
SEQUENCE: 256
titcgagaggg ttaaatcaaa t
SEO ID NO 257 LENGTH: 21 TYPE: DNA
ORGANISM: Artificial Sequence FEATURE:
OTHER INFORMATION: Sense primer
SEQUENCE: 257
caaatctgat gtcggtgagg a
SEO ID NO 258 LENGTH 19 TYPE: DNA
ORGANISM: Artificial Sequence FEATURE:
OTHER INFORMATION: Sense primer
SEQUENCE: 258
taaatcaaat citgatgtcg
SEO ID NO 259 LENGTH: 21 TYPE: DNA
ORGANISM: Artificial Sequence FEATURE:
OTHER INFORMATION: Sense primer
SEQUENCE: 259
gagagggitta aatcaaatct g
SEQ ID NO 260 LENGTH: 21 TYPE: DNA
66
- Continued
21
19
21
21
19
21
Jun. 9, 2011
US 2011/O 1361.25 A1
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Sense primer
<4 OOs, SEQUENCE: 260
atctgatgtc. g.gtgaggaggg
<210s, SEQ ID NO 261 &211s LENGTH: 19 &212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Sense primer
<4 OOs, SEQUENCE: 261
aatticgagag ggittaaatc
<210s, SEQ ID NO 262 &211s LENGTH: 19 &212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Sense primer
<4 OOs, SEQUENCE: 262
gatgtcggtg aggagggitt
<210s, SEQ ID NO 263 &211s LENGTH: 19 &212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Sense primer
<4 OOs, SEQUENCE: 263
gagggatggc agtgtcgga
<210s, SEQ ID NO 264 &211s LENGTH: 19 &212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Sense primer
<4 OOs, SEQUENCE: 264
tggccaagac ttittct cat
<210s, SEQ ID NO 265 &211s LENGTH: 2O &212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Sense primer
<4 OOs, SEQUENCE: 265
gatgagtctg caacctatica
<210s, SEQ ID NO 266 &211s LENGTH: 21 &212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Sense primer
67
- Continued
21
19
19
19
19
Jun. 9, 2011
US 2011/O 1361.25 A1
<4 OOs, SEQUENCE: 266
tagcagagag gtggtgcatgg
<210s, SEQ ID NO 267 &211s LENGTH: 21 &212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Sense primer
<4 OOs, SEQUENCE: 267
acggcc actg citat caagtt c
<210s, SEQ ID NO 268 &211s LENGTH: 2O &212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Antisense primer
<4 OOs, SEQUENCE: 268
aagctcgt.ca ggttgact ct
<210s, SEQ ID NO 269 &211s LENGTH: 19 &212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Antisense primer
<4 OOs, SEQUENCE: 269
gtaa.gctcgt caggttgac
<210s, SEQ ID NO 270 &211s LENGTH: 19 &212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Antisense primer
<4 OOs, SEQUENCE: 270
cgagtaagct cqt caggtt
<210s, SEQ ID NO 271 &211s LENGTH: 19 &212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Antisense primer
<4 OOs, SEQUENCE: 271
Ctgctatcga gtaagctcg
<210s, SEQ ID NO 272 &211s LENGTH: 19 &212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Antisense primer
<4 OOs, SEQUENCE: 272
titta accctic ticgaattaa
68
- Continued
21
21
19
19
19
19
Jun. 9, 2011
US 2011/O 1361.25 A1
<210s, SEQ ID NO 273 &211s LENGTH: 19 &212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Antisense primer
<4 OOs, SEQUENCE: 273
citcc togctat cqagtaagc
<210s, SEQ ID NO 274 &211s LENGTH: 2O &212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Antisense primer
<4 OOs, SEQUENCE: 274
t cagatttga tittaac cct c
<210s, SEQ ID NO 275 &211s LENGTH: 2O &212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Antisense primer
< 400 SEQUENCE: 275
accctic ct ca ccgacat cag
<210s, SEQ ID NO 276 &211s LENGTH: 19 &212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Antisense primer
<4 OOs, SEQUENCE: 276
acat cagatt tdatttaac
<210s, SEQ ID NO 277 &211s LENGTH: 19 &212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Antisense primer
<4 OO > SEQUENCE: 277
ccga catcag atttgattit
<210s, SEQ ID NO 278 &211s LENGTH: 19 &212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Antisense primer
<4 OO > SEQUENCE: 278
tgatttaa.cc ct citcgaat
<210s, SEQ ID NO 279 &211s LENGTH: 19 &212s. TYPE: DNA
69
- Continued
19
19
19
19
Jun. 9, 2011
US 2011/O 1361.25 A1
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Antisense primer
<4 OO > SEQUENCE: 279
t caccgacat cagatttga
<210s, SEQ ID NO 280 &211s LENGTH: 19 &212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Antisense primer
<4 OOs, SEQUENCE: 280
atttgattta accct citcg
<210s, SEQ ID NO 281 &211s LENGTH: 21 &212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Antisense primer
<4 OOs, SEQUENCE: 281
Ctacctgata ggttgcagac t
<210s, SEQ ID NO 282 &211s LENGTH: 2O &212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Antisense primer
<4 OOs, SEQUENCE: 282
gcaccacctic tictogctat cq
<210s, SEQ ID NO 283 &211s LENGTH: 19 &212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Antisense primer
<4 OOs, SEQUENCE: 283
atcc ct caac ggaaaag.ca
<210s, SEQ ID NO 284 &211s LENGTH: 2O &212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Antisense primer
<4 OOs, SEQUENCE: 284
acacttaaag togaacgcc ct
<210s, SEQ ID NO 285 &211s LENGTH: 19 &212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Antisense primer
70
- Continued
19
19
21
19
Jun. 9, 2011
US 2011/O 1361.25 A1
<4 OOs, SEQUENCE: 285
tcqctic cqac act gccatc
<210s, SEQ ID NO 286 &211s LENGTH: 2O &212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Antisense primer
<4 OOs, SEQUENCE: 286
ccgatctgat gtc.ttgcagt
<210s, SEQ ID NO 287 &211s LENGTH: 19 &212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Antisense primer
<4 OO > SEQUENCE: 287
atgagaaaag ticttggc.ca
<210s, SEQ ID NO 288 &211s LENGTH: 19 &212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Sense primer
<4 OOs, SEQUENCE: 288
agggcgttac C cctagtgc
<210s, SEQ ID NO 289 &211s LENGTH: 19 &212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Sense primer
<4 OOs, SEQUENCE: 289
tacic cc tagt gcc ct cqca
<210s, SEQ ID NO 290 &211s LENGTH: 21 &212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Sense primer
<4 OOs, SEQUENCE: 290
gcgc.ccgtag ccggcctgta a
<210s, SEQ ID NO 291 &211s LENGTH: 21 &212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Sense primer
<4 OOs, SEQUENCE: 291
gaggttct Co. tcc.gcgagggg
71
- Continued
19
19
19
19
21
21
Jun. 9, 2011
US 2011/O 1361.25 A1
SEQ ID NO 292 LENGTH: 21 TYPE: DNA
ORGANISM: Artificial Sequence FEATURE:
OTHER INFORMATION: Sense primer
SEQUENCE: 292
gcaccaggcg cggaacgt.cc C
SEQ ID NO 293 LENGTH: 2O TYPE: DNA
ORGANISM: Artificial Sequence FEATURE:
OTHER INFORMATION: Sense primer
SEQUENCE: 293
gaggtogagc titct cotc.cg
SEQ ID NO 294 LENGTH 19 TYPE: DNA
ORGANISM: Artificial Sequence FEATURE:
OTHER INFORMATION: Sense primer
SEQUENCE: 294
c cct agtgcc ct cqcaaga
SEO ID NO 295 LENGTH: 21 TYPE: DNA
ORGANISM: Artificial Sequence FEATURE:
OTHER INFORMATION: Sense primer
SEQUENCE: 295
cc.cgtagc.cg gcctgtaaag t
SEQ ID NO 296 LENGTH: 21 TYPE: DNA
ORGANISM: Artificial Sequence FEATURE:
OTHER INFORMATION: Sense primer
SEQUENCE: 296
cggggtggga gatcgagctt C
SEO ID NO 297 LENGTH: 2O TYPE: DNA
ORGANISM: Artificial Sequence FEATURE:
OTHER INFORMATION: Sense primer
SEQUENCE: 297
gtcgagct tc. tcct cogcga
SEQ ID NO 298 LENGTH: 21 TYPE: DNA
72
- Continued
21
19
21
21
Jun. 9, 2011
US 2011/O 1361.25 A1
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Sense primer
<4 OOs, SEQUENCE: 298
ggtgggagglt c.gagct tctic C
<210s, SEQ ID NO 299 &211s LENGTH: 19 &212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Sense primer
<4 OOs, SEQUENCE: 299
tCggggtggg aggtoga.gc
<210s, SEQ ID NO 3 OO &211s LENGTH: 19 &212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Sense primer
<4 OOs, SEQUENCE: 3 OO
gcqttacc cc tagtgc cct
<210s, SEQ ID NO 3 O1 &211s LENGTH: 21 &212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Sense primer
<4 OOs, SEQUENCE: 301
tagggg tagg gctaagc.cat g
<210s, SEQ ID NO 3 O2 &211s LENGTH: 19 &212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Sense primer
<4 OOs, SEQUENCE: 302
cgcaccaggc gcggaacgt.
<210s, SEQ ID NO 3 O3 &211s LENGTH: 19 &212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Sense primer
<4 OOs, SEQUENCE: 303
gggaggtoga gct tct cot
<210s, SEQ ID NO 3 O4 &211s LENGTH: 2O &212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Sense primer
73
- Continued
21
19
19
21
19
19
Jun. 9, 2011
US 2011/O 1361.25 A1
<4 OOs, SEQUENCE: 304
aggtggagga ataagcgggg
<210s, SEQ ID NO 3 O5 &211s LENGTH: 19 &212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Sense primer
<4 OOs, SEQUENCE: 305
gaaaggtgga ggaataagc
<210s, SEQ ID NO 3 O6 &211s LENGTH: 21 &212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Sense primer
<4 OOs, SEQUENCE: 306
gggagt cqta cqct ct cqgg a
<210s, SEQ ID NO 3 O7 &211s LENGTH: 21 &212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Sense primer
<4 OO > SEQUENCE: 3 O7
ctaacctgcc cittgggat.ct g
<210s, SEQ ID NO 3 O8 &211s LENGTH: 19 &212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Antisense primer
<4 OOs, SEQUENCE: 308
ggcact aggg gtaacgc.cc
<210s, SEQ ID NO 3 O9 &211s LENGTH: 2O &212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Antisense primer
<4 OOs, SEQUENCE: 309
agaa.gct cqa cct cocaccc
<210s, SEQ ID NO 310 &211s LENGTH: 2O &212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Antisense primer
<4 OOs, SEQUENCE: 310
tacaggc.cgg ctacgggcgc
74
- Continued
19
21
21
19
Jun. 9, 2011
US 2011/O 1361.25 A1
SEQ ID NO 311 LENGTH 19 TYPE: DNA
ORGANISM: Artificial Sequence FEATURE:
OTHER INFORMATION: Antisense primer
SEQUENCE: 311
agct cq acct c ccaccc.cg
SEQ ID NO 312 LENGTH 19 TYPE: DNA
ORGANISM: Artificial Sequence FEATURE:
OTHER INFORMATION: Antisense primer
SEQUENCE: 312
CCCCtcgcgg aggaga agc
SEQ ID NO 313 LENGTH 19 TYPE: DNA
ORGANISM: Artificial Sequence FEATURE:
OTHER INFORMATION: Antisense primer
SEQUENCE: 313
tgcgagggca Ctaggggta
SEQ ID NO 314 LENGTH: 21 TYPE: DNA
ORGANISM: Artificial Sequence FEATURE:
OTHER INFORMATION: Antisense primer
SEQUENCE: 314
tgactttaca ggc.cggctac g
SEO ID NO 315 LENGTH: 21 TYPE: DNA
ORGANISM: Artificial Sequence FEATURE:
OTHER INFORMATION: Antisense primer
SEQUENCE: 315
catggct tag ccctaccc ct a
SEQ ID NO 316 LENGTH: 2O TYPE: DNA
ORGANISM: Artificial Sequence FEATURE:
OTHER INFORMATION: Antisense primer
SEQUENCE: 316
aggaga agct cacct coca
SEO ID NO 317 LENGTH 19 TYPE: DNA
75
- Continued
19
19
19
21
21
Jun. 9, 2011
US 2011/O 1361.25 A1
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Antisense primer
<4 OOs, SEQUENCE: 317
gacgttcc.gc gcc tdgtgc
<210s, SEQ ID NO 318 &211s LENGTH: 2O &212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Antisense primer
<4 OOs, SEQUENCE: 318
Ctttac aggc cqgctacggg
<210s, SEQ ID NO 319 &211s LENGTH: 19 &212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Antisense primer
<4 OOs, SEQUENCE: 319
tcttgcgagg gCactaggg
<210s, SEQ ID NO 320 &211s LENGTH: 2O &212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Antisense primer
<4 OOs, SEQUENCE: 32O
cggaggagaa gct cacctic
<210s, SEQ ID NO 321 &211s LENGTH: 2O &212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Antisense primer
<4 OOs, SEQUENCE: 321
tcgcggagga gaagct cac
<210s, SEQ ID NO 322 &211s LENGTH: 19 &212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Antisense primer
<4 OOs, SEQUENCE: 322
gagggc act a giggtaacg
<210s, SEQ ID NO 323 &211s LENGTH: 2O &212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Antisense primer
76
- Continued
19
19
19
Jun. 9, 2011
US 2011/O 1361.25 A1
SEQUENCE: 323
accc.cgaggg gcaa.gaggcc
SEQ ID NO 324 LENGTH: 21 TYPE: DNA
ORGANISM: Artificial Sequence FEATURE:
OTHER INFORMATION: Antisense primer
SEQUENCE: 324
ggggittatcc agat.cccaag g
SEO ID NO 325 LENGTH: 2O TYPE: DNA
ORGANISM: Artificial Sequence FEATURE:
OTHER INFORMATION: Antisense primer
SEQUENCE: 325
gccacgcc ct citt cocq aga
SEQ ID NO 326 LENGTH: 2O TYPE: DNA
ORGANISM: Artificial Sequence FEATURE:
OTHER INFORMATION: Antisense primer
SEQUENCE: 326
gttatccaga t cc caagggc
SEO ID NO 327 LENGTH: 21 TYPE: DNA
ORGANISM: Artificial Sequence FEATURE:
OTHER INFORMATION: Antisense primer
SEQUENCE: 327
cittatt cotc. cacctittctg g
SEQ ID NO 328 LENGTH 19 TYPE: DNA
ORGANISM: Artificial Sequence FEATURE:
OTHER INFORMATION: Sense primer
SEQUENCE: 328
taalacc ctgc cqcagttgg
SEQ ID NO 329 LENGTH 19 TYPE: DNA
ORGANISM: Artificial Sequence FEATURE:
OTHER INFORMATION: Sense primer
SEQUENCE: 329
ccittaaac cc togcc.gcagt
77
- Continued
21
21
19
19
Jun. 9, 2011
US 2011/O 1361.25 A1
<210s, SEQ ID NO 330 &211s LENGTH: 19 &212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Sense primer
<4 OOs, SEQUENCE: 330
gtcCtggaac ggttcCtcg
<210s, SEQ ID NO 331 &211s LENGTH: 21 &212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Sense primer
<4 OOs, SEQUENCE: 331
Ctct acaaag gcgggggaat a
<210s, SEQ ID NO 332 &211s LENGTH: 2O &212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Sense primer
< 4 OO > SEQUENCE: 332
Ctggaacggit to Ctcgctga
<210s, SEQ ID NO 333 &211s LENGTH: 21 &212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Sense primer
<4 OOs, SEQUENCE: 333
ggcgaggagt CCtggaacgg t
<210s, SEQ ID NO 334 &211s LENGTH: 19 &212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Sense primer
<4 OOs, SEQUENCE: 334
titt.ccc.cgct c tacaaagg
<210s, SEQ ID NO 335 &211s LENGTH: 21 &212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Sense primer
<4 OOs, SEQUENCE: 335
tacaaaggcg ggggaataag C
<210s, SEQ ID NO 336 &211s LENGTH: 19 &212s. TYPE: DNA
78
- Continued
19
21
21
19
21
Jun. 9, 2011
US 2011/O 1361.25 A1
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Sense primer
<4 OOs, SEQUENCE: 336
cgct ct acaa aggcggggg
<210s, SEQ ID NO 337 &211s LENGTH: 2O &212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Sense primer
<4 OO > SEQUENCE: 337
at aggcgagg agt cctggaa
<210s, SEQ ID NO 338 &211s LENGTH: 19 &212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Sense primer
<4 OOs, SEQUENCE: 338
c cat aggcga ggagtic ctg
<210s, SEQ ID NO 339 &211s LENGTH: 19 &212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Sense primer
<4 OOs, SEQUENCE: 339
gcttitt cocc gct ctacaa
<210s, SEQ ID NO 340 &211s LENGTH: 2O &212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Sense primer
<4 OOs, SEQUENCE: 340
gcta acctac Cctgaggagg
<210s, SEQ ID NO 341 &211s LENGTH: 2O &212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Sense primer
<4 OOs, SEQUENCE: 341
tctic ccatag gcgaggagtic
<210s, SEQ ID NO 342 &211s LENGTH: 19 &212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Sense primer
79
- Continued
19
19
19
Jun. 9, 2011
US 2011/O 1361.25 A1
<4 OOs, SEQUENCE: 342
tggcta acct accCtgagg
<210s, SEQ ID NO 343 &211s LENGTH: 19 &212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Sense primer
<4 OOs, SEQUENCE: 343
ataatcto cc at aggcgag
<210s, SEQ ID NO 344 &211s LENGTH: 2O &212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Sense primer
<4 OOs, SEQUENCE: 344
tgaggaggga gata accc.cg
<210s, SEQ ID NO 345 &211s LENGTH: 21 &212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Sense primer
<4 OOs, SEQUENCE: 345
acacgtggct aacct accct g
<210s, SEQ ID NO 346 &211s LENGTH: 19 &212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Sense primer
<4 OOs, SEQUENCE: 346
Cctgaggagg gagatalacc
<210s, SEQ ID NO 347 &211s LENGTH: 19 &212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Sense primer
<4 OOs, SEQUENCE: 347
aaactgggga taatct coc
<210s, SEQ ID NO 348 &211s LENGTH: 19 &212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Antisense primer
<4 OOs, SEQUENCE: 348
cCaactg.cgg Cagggittta
80
- Continued
19
19
21
19
19
19
Jun. 9, 2011
US 2011/O 1361.25 A1
<210s, SEQ ID NO 349 &211s LENGTH: 19 &212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Antisense primer
<4 OOs, SEQUENCE: 349
actg.cggcag ggtttalagg
<210s, SEQ ID NO 350 &211s LENGTH: 21 &212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Antisense primer
<4 OOs, SEQUENCE: 350
cgagga accg titcCaggact c
<210s, SEQ ID NO 351 &211s LENGTH: 2O &212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Antisense primer
< 4 OO > SEQUENCE: 351
aaccottcca ggactic ct cq
<210s, SEQ ID NO 352 &211s LENGTH: 21 &212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Antisense primer
<4 OOs, SEQUENCE: 352
tccaggactic ct cqcctatgg
<210s, SEQ ID NO 353 &211s LENGTH: 19 &212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Antisense primer
<4 OOs, SEQUENCE: 353
CCtttgtaga gcggggaaa
<210s, SEQ ID NO 3.54 &211s LENGTH: 2O &212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Antisense primer
<4 OOs, SEQUENCE: 354
agcgaggaac C9ttcCagga
<210s, SEQ ID NO 355 &211s LENGTH: 21 &212s. TYPE: DNA
81
- Continued
19
21
21
19
Jun. 9, 2011
US 2011/O 1361.25 A1
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Antisense primer
<4 OO > SEQUENCE: 355
cgttccagga citcct cqcct a
<210s, SEQ ID NO 356 &211s LENGTH: 19 &212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Antisense primer
<4 OOs, SEQUENCE: 356
CCCCCCCtt ttagagcg
<210s, SEQ ID NO 357 &211s LENGTH: 2O &212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Antisense primer
<4 OO > SEQUENCE: 357
ttcagcgagg aaccgttc.ca
<210s, SEQ ID NO 358 &211s LENGTH: 19 &212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Antisense primer
<4 OOs, SEQUENCE: 358
attic cc cc.gc citttgtaga
<210s, SEQ ID NO 359 &211s LENGTH: 19 &212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Antisense primer
<4 OO > SEQUENCE: 359
ttgtagagcg gggaaaagc
<210s, SEQ ID NO 360 &211s LENGTH: 21 &212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Antisense primer
<4 OOs, SEQUENCE: 360
atct coct co toaggg tagg t
<210s, SEQ ID NO 3 61 &211s LENGTH: 19 &212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Antisense primer
82
- Continued
21
19
19
19
21
Jun. 9, 2011
US 2011/O 1361.25 A1
SEQUENCE: 361
gggittatcto cotcct cag
SEQ ID NO 362 LENGTH 19 TYPE: DNA
ORGANISM: Artificial Sequence FEATURE:
OTHER INFORMATION: Antisense primer
SEQUENCE: 362
tcgc.ctatgg gagattatic
SEQ ID NO 363 LENGTH: 2O TYPE: DNA
ORGANISM: Artificial Sequence FEATURE:
OTHER INFORMATION: Antisense primer
SEQUENCE: 363
t caggg tagg ttagcc acgt.
SEQ ID NO 364 LENGTH 19 TYPE: DNA
ORGANISM: Artificial Sequence FEATURE:
OTHER INFORMATION: Antisense primer
SEQUENCE: 364
Ccticaggg taggittagc.ca
SEO ID NO 365 LENGTH 19 TYPE: DNA
ORGANISM: Artificial Sequence FEATURE:
OTHER INFORMATION: Antisense primer
SEQUENCE: 365
ccggggittat citc cct cot
SEQ ID NO 366 LENGTH 19 TYPE: DNA
ORGANISM: Artificial Sequence FEATURE:
OTHER INFORMATION: Antisense primer
SEQUENCE: 366
t cct cqccta tiggagatt
SEO ID NO 367 LENGTH 19 TYPE: DNA
ORGANISM: Artificial Sequence FEATURE:
OTHER INFORMATION: Antisense primer
SEQUENCE: 367
Cctic ct cagg gtaggittag
83
- Continued
19
19
19
19
19
19
Jun. 9, 2011
US 2011/O 1361.25 A1
SEQ ID NO 368 LENGTH: 2O TYPE: DNA
ORGANISM: Artificial Sequence FEATURE:
OTHER INFORMATION: Sense primer
SEQUENCE: 368
t cctgaaagg acgaccggtg
SEO ID NO 369 LENGTH 19 TYPE: DNA
ORGANISM: Artificial Sequence FEATURE:
OTHER INFORMATION: Sense primer
SEQUENCE: 369
ggactgaggg Ctgtaactic
SEO ID NO 37O LENGTH: 21 TYPE: DNA
ORGANISM: Artificial Sequence FEATURE:
OTHER INFORMATION: Sense primer
SEQUENCE: 370
gaggttgaat gtactitt Cagg
SEO ID NO 371 LENGTH: 2O TYPE: DNA
ORGANISM: Artificial Sequence FEATURE:
OTHER INFORMATION: Sense primer
SEQUENCE: 371
ggtggcgaaa gogttcaact
SEO ID NO 372 LENGTH 19 TYPE: DNA
ORGANISM: Artificial Sequence FEATURE:
OTHER INFORMATION: Sense primer
SEQUENCE: 372
gccCtcacga atgtggatt
SEO ID NO 373 LENGTH: 2O TYPE: DNA
ORGANISM: Artificial Sequence FEATURE:
OTHER INFORMATION: Sense primer
SEQUENCE: 373
acct cqaaac ccgttcgtag
SEO ID NO 374 LENGTH 19 TYPE: DNA
84
- Continued
19
21
19
Jun. 9, 2011
US 2011/O 1361.25 A1
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Sense primer
<4 OOs, SEQUENCE: 374
tcc.gtag taa ticgtaggit c
<210s, SEQ ID NO 375 &211s LENGTH: 21 &212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Sense primer
<4 OO > SEQUENCE: 375
atcc td taat cotgaaagga c
<210s, SEQ ID NO 376 &211s LENGTH: 2O &212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Sense primer
<4 OO > SEQUENCE: 376
gtag to agga citgagggctg
<210s, SEQ ID NO 377 &211s LENGTH: 21 &212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Sense primer
<4 OO > SEQUENCE: 377
aggacga.ccg gtggcgaaag C
<210s, SEQ ID NO 378 &211s LENGTH: 2O &212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Sense primer
<4 OO > SEQUENCE: 378
taactic gocc ticacgaatgt
<210s, SEQ ID NO 379 &211s LENGTH: 19 &212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Sense primer
<4 OO > SEQUENCE: 379
gaaggtgtta agtgggtca
<210s, SEQ ID NO 38O &211s LENGTH: 21 &212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Sense primer
85
- Continued
19
21
21
19
Jun. 9, 2011
US 2011/O 1361.25 A1
SEQUENCE: 38O
aalaccc.gttc gtagt cagga C
SEQ ID NO 381 LENGTH: 2O TYPE: DNA
ORGANISM: Artificial Sequence FEATURE:
OTHER INFORMATION: Sense primer
SEQUENCE: 381
tacggtgaat atgc.ccctgc
SEQ ID NO 382 LENGTH: 21 TYPE: DNA
ORGANISM: Artificial Sequence FEATURE:
OTHER INFORMATION: Sense primer
SEQUENCE: 382
cacttggtgt togcttctic cq t
SEQ ID NO 383 LENGTH 19 TYPE: DNA
ORGANISM: Artificial Sequence FEATURE:
OTHER INFORMATION: Sense primer
SEQUENCE: 383
gat cacttitt attgagtict
SEQ ID NO 384 LENGTH: 2O TYPE: DNA
ORGANISM: Artificial Sequence FEATURE:
OTHER INFORMATION: Sense primer
SEQUENCE: 384
agcatcagga ataagggctg
SEO ID NO 385 LENGTH 19 TYPE: DNA
ORGANISM: Artificial Sequence FEATURE:
OTHER INFORMATION: Sense primer
SEQUENCE: 385
aaga cc cc catctictaatt
SEQ ID NO 386 LENGTH 19 TYPE: DNA
ORGANISM: Artificial Sequence FEATURE:
OTHER INFORMATION: Sense primer
SEQUENCE: 386
ccggtott at aaatcttca
86
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SEO ID NO 387 LENGTH: 2O TYPE: DNA
ORGANISM: Artificial Sequence FEATURE:
OTHER INFORMATION: Sense primer
SEQUENCE: 387
ataacgagca agaccc cc at
SEQ ID NO 388 LENGTH: 2O TYPE: DNA
ORGANISM: Artificial Sequence FEATURE:
OTHER INFORMATION: Antisense primer
SEQUENCE: 388
Caggggcata t t caccgtag
SEO ID NO 389 LENGTH 19 TYPE: DNA
ORGANISM: Artificial Sequence FEATURE:
OTHER INFORMATION: Antisense primer
SEQUENCE: 389
t caggattac aggattitta
SEO ID NO 390 LENGTH: 21 TYPE: DNA
ORGANISM: Artificial Sequence FEATURE:
OTHER INFORMATION: Antisense primer
SEQUENCE: 390
accctgaaag tacattcaac c
SEQ ID NO 391 LENGTH 19 TYPE: DNA
ORGANISM: Artificial Sequence FEATURE:
OTHER INFORMATION: Antisense primer
SEQUENCE: 391
gccaccggtc gtc.ctitt.ca
SEQ ID NO 392 LENGTH 19 TYPE: DNA
ORGANISM: Artificial Sequence FEATURE:
OTHER INFORMATION: Antisense primer
SEQUENCE: 392
c tagttgaac gctitt.cgc.c
SEO ID NO 393 LENGTH: 21 TYPE: DNA
87
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<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Antisense primer
<4 OOs, SEQUENCE: 393
tcqtcc titt.c aggattacag g
<210s, SEQ ID NO 394 &211s LENGTH: 21 &212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Antisense primer
<4 OOs, SEQUENCE: 394
acgctitt.cgc caccggtcgt c
<210s, SEQ ID NO 395 &211s LENGTH: 19 &212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Antisense primer
<4 OO > SEQUENCE: 395
gggttt cag gttagctt C
<210s, SEQ ID NO 396 &211s LENGTH: 19 &212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Antisense primer
<4 OOs, SEQUENCE: 396
c cct cagt cc tdactacga
<210s, SEQ ID NO 397 &211s LENGTH: 2O &212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Antisense primer
<4 OO > SEQUENCE: 397
Ctgaagattt atalagaccgg
<210s, SEQ ID NO 398 &211s LENGTH: 21 &212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Antisense primer
<4 OOs, SEQUENCE: 398
ttacagcc ct cagtcc togac t
<210s, SEQ ID NO 399 &211s LENGTH: 21 &212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Antisense primer
88
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<4 OOs, SEQUENCE: 399
aatcCacatt C9tgagggcg a
<210s, SEQ ID NO 4 OO &211s LENGTH: 2O &212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Antisense primer
<4 OOs, SEQUENCE: 4 OO
atgggggtct tctcgtt at
<210s, SEQ ID NO 4O1 &211s LENGTH: 19 &212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Antisense primer
<4 OOs, SEQUENCE: 4 O1
gctgttgacc tacgattac
<210s, SEQ ID NO 4 O2 &211s LENGTH: 21 &212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Antisense primer
<4 OOs, SEQUENCE: 4 O2
ccitacgatta citacggaatc c
<210s, SEQ ID NO 403 &211s LENGTH: 19 &212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Antisense primer
<4 OOs, SEQUENCE: 403
acccacttaa caccitt cqc
<210s, SEQ ID NO 404 &211s LENGTH: 2O &212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Antisense primer
<4 OOs, SEQUENCE: 404
cc caagttctt acagt ct citt
<210s, SEQ ID NO 405 &211s LENGTH: 2O &212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Antisense primer
<4 OOs, SEQUENCE: 405
ctaccctgaaagtacattca
89
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- Continued
<210s, SEQ ID NO 4O6 &211s LENGTH: 19 &212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Antisense primer
<4 OOs, SEQUENCE: 406
cago cott at tcc tdatgc 19
<210s, SEQ ID NO 4O7 &211s LENGTH: 2O &212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: Antisense primer
<4 OOs, SEQUENCE: 4 O7
ggtcgt.cct t t caggattac 2O
<210s, SEQ ID NO 408 &211s LENGTH: 18 &212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: PCR primer
< 4 OO > SEQUENCE: 408
gtgc.ca.gcmg cc.gcggta 18
<210s, SEQ ID NO 409 &211s LENGTH: 17 &212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: PCR primer
<4 OOs, SEQUENCE: 409
Cctacgggag gcagoag 17
<210s, SEQ ID NO 410 &211s LENGTH: 19 &212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: PCR primer
<4 OOs, SEQUENCE: 410
cc.gt caattic ctittgagtt 19
<210s, SEQ ID NO 411 &211s LENGTH: 18 &212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: PCR primer
<4 OOs, SEQUENCE: 411
gctgcctic cc gtaggagt 18
<210s, SEQ ID NO 412 &211s LENGTH: 18 &212s. TYPE: DNA
US 2011/O 1361.25 A1
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: PCR primer
<4 OOs, SEQUENCE: 412
gggcggWgtg tacaaggc
<210s, SEQ ID NO 413 &211s LENGTH: 2O
&212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: PCR primer
<4 OOs, SEQUENCE: 413
acgggg.cgca gCaggcgcga
<210s, SEQ ID NO 414 &211s LENGTH: 19
&212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: PCR primer
<4 OOs, SEQUENCE: 414
gtgc.ca.gcag cc.gcggtaa
<210s, SEQ ID NO 415 &211s LENGTH: 19
&212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: PCR primer
<4 OOs, SEQUENCE: 415
yccgg.cgttg amt coaatt
<210s, SEQ ID NO 416 &211s LENGTH: 2O
&212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: PCR primer
<4 OOs, SEQUENCE: 416
gtgctic cc cc gccaattic ct
<210s, SEQ ID NO 417 &211s LENGTH: 17
&212s. TYPE: DNA
<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:
<223> OTHER INFORMATION: PCR primer
<4 OOs, SEQUENCE: 417
attaccgcgg Ctgctgg
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What is claimed is: 1. Method to identify and quantify environmental micro
organisms useful in biomining processes, wherein said method comprises the steps of
(a) extracting DNA from a sample: (b) quantifying the extracted DNA; (c) performing a quantitative PCR (qPCR) technique,
using either said DNA sample, and specific primers of SEQ ID No. 4 to SEQID No. 407 for each taxon to be determined, where taxons are selected from: i. Bacteria: Total bacteria, Acidiphilium sp., Leptospir
illum sp., Sulfobacillus sp., Acidithiobacillus fer rooxidans and Acidithiobacillus thiooxidans, and
ii. Archaea: Total archaea, Acidianus sp., Ferroplasma sp, Metallosphaera sp., Sulfolobus sp. and Thermo plasma sp.,
(d) calculating the number of microorganisms in the sample that belong to each of the analyzed taxons according to formula (3)
92 Jun. 9, 2011
N QN molecules *Tng (3) Ceit (mL)-(g) Cmolecules/cel * Ung * Cn(mL)-(g)
wherein:
N=Estimated Concentration of microorganisms. Q=Number of Molecules as calculated with the standard
curve using the C(T) value given by the equipment. T=Total DNA isolated from original sample. U=Amount of DNA used in the qPCR reaction. C=Number of molecules or copies by genome of the spe
cific DNA fragment detected. Cm=Amount in ''g'' or “ml of the processed sample.
c c c c c