PCR Brochure 2010

MP Biomedicals Europe • tel : 00800 7777 9999 • fax : 00800 6666 8888

ISIS™

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MP Biomedicals PCR Line

YOU… Your PCR… Your Time and OUR Solutions

Wide & Convenient PCR Range

Multiplex PCR

Isis™ High Fidelity

Optimizing your PCR?Take your time to choose the right product!

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High Quality Taq DNA polymeraseMP Biomedicals (formerly Qbiogene) has manufactured thermostable polymerases for 15 years. This collective expertise ensures:

• Lot-to-lot reproducibilityThe strict quality control procedure guarantees the exact enzyme activity for each produced batch. It also includes control of contaminants like nickases, endo/exonucleases, ribonu-cleases, and bacterial/plasmid DNA.

• Results reliability with ultra-pure polymeraseTaq DNA polymerase is highly purified in order to obtain the lowest possible contamination from E. coli DNA or plasmid DNA and then avoid PCR-false positives results.

• Complete Buffer SystemThe unique 10x reaction buffer has been optimized for maximum stability and efficiency in any PCR reaction. For particular applications, other buffers systems are available.

• Broad range of pack size and convenient Mastermix or Core Kit formatThe wide choice of pack size, buffers and enzyme concentrations meets the need of every PCR application. Moreover, Core Kit provides with all the reagents necessary to amplify, excepted template and primers.

• High amplification yieldPerformances comparison with other Taq DNA polymerase sources showed comparable or superior amplification yield.

How to overcome PCR-false positives?β-Lactamases produced by bacterium are known to protect against the lethal effect of β-lactam antibiotics. Extended-spectrum β-lactamases are often plasmid-mediated, and most of them are mutants of the classic TEM- and SHV-type enzymes. The blaTEM-1a gene has been the most commonly used selective marker for expression vectors that are generally presented in multiple copies like pBR322 plasmid. Thus, it is likely that during Taq DNA polymerase purification, the DNA harbouring blaTEM-1a gene was not completely removed and thus producing PCR-false positive when primers can match this contaminant, like cloning or antibiotic resistance assays.

Song and co-workers describe in The Journal of Microbiology, February 2006, 44(1):126-128 a method to decontaminate commercial Taq DNA polymerase by DNase I incubation followed by carefully DNase I inactivation.

Second possibility to overcome false positives: use highly purified Taq DNA polymerase without exogenous DNA con-tamination. Koncan et al, in Journal of Antimicrobial Chemotherapy, September 2007 ; 60(3) : 702-703 compared purity of two commercial available polymerases by validation of PCR results with a second technique: antibiotic Hydrolysis assay on crude extract of isolates.

“My experience shows that caution is required to the degree of Taq DNA polymerase purity when bacteria antibiotic-resistance is tested by PCR. (1)

I have adopted MP Biomedicals (formerly Qbiogene) Taq-Core polymerase because its high-purity ensures more reliable results.”

Rosa Mª del Campo Moreno.Hospital Ramon y Cajal MADRID - SPAIN(1)Journal of Antimicrobial Chemotherapy 2007 Sept ; 60(3):702-3

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Complete Buffer system:MP Biomedicals high purified, recombinant, Taq DNA polymerase is supplied with buffers for each application, at two different concentrations and convenient pack size.

• Standard Buffer: well known QBiogene 10x Incubation PCR Buffer with MgCl2 at 1,5 mM as final concentration. Used since years by researchers in a broad range of reaction conditions.

• XD Buffer: without detergent and without BSA, particularly suitable for automated reaction set up or HPLC systems. Other special applications are PCR on vegetable or beef material.

• (NH4)2SO4 Buffer: it has been reported that buffers containing Ammonium Sulphate are able to improve the efficiency and specificity of some amplifications.

• Direct Loading Buffer: a PCR Buffer which contains a densifying agent and red purple dye that allows direct loading amplifications after the cycling without new handle.

For most applications, simply choice your Taq DNA polymerase concentration, the optimal buffer and dNTPs of high degree purity with the well know MP Biomedicals quality at www.mpbio.com

Cat # Size Tubes x UTaq DNA pol

ConcentrationBuffer Included

EPTQA025 1x250 U

5 U/µl 10x Standard Buffer with Mg10x Standard Buffer without Mg25 mM MgCl2

EPTQA325 3x250 U

EPTQA925 10x250 U

EPTQA100 1x1000 U

EPTQA105 5x1000 U

EPTQA125 25x1000 U

EPTQA500 1x5000 U

EPTQC060 1x600 U15 U/µl

EPTQC200 1x2000 U

EPTQD025 1x250 U

5 U/µl10x Standard Buffer with MgEPTQD325 3x250 U

EPTQD925 10x250 U

EPTQD105 5x1000 U

EPTQX025 1x250 U

5 U/µl10x XD Buffer with Mg

EPTQX325 3x250 U

EPTQX925 10x250 U

EPTQX100 1000 U

EPTQX060 1x600 U15 U/µl

EPTQX200 1x2000 U

EPTQS001 1x250 U5 U/µl

10x (NH4)2SO4 Buffer without Mg50 mM MgCl2EPTQS901 10x250 U

EPTQL025 1x250 U5 U/µl

5x Direct Loading Buffer Buffer with Mg 25mM MgCI2EPTQL100 1x1000 U

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2U

Taq CORE kits Concept MP Biomedicals offers the opportunity to improve your workflow providing with all the necessary to perform PCR in one pack. Taq CORE kits contain all the reagents (presented as separate items) required for amplification:

> High quality recombinant Taq DNA polymerase

> High purity dNTPs Mix at either 10 mM or 25 mM of each dNTP

> Optimized incubation buffers with/without Mg

> Separate MgCl2 solution.

CORE kit Components

Cat #

SizeWith dNTPs10 mM each

With dNTPs25 mM each

Taq DNA Polymerase 5 U/µl 10 x Standard Buffer with Mg10x Standard Buffer without Mg 25 mM MgCl2dNTPs mix

1x250 U EPTQK101 EPTQK251


5x250 U EPTQK105C EPTQK255C


1x1000 U EPTQK300

5x1000 U EPTQK10L EPTQK25L

Taq DNA Polymerase 15 U/µl10x Standard Buffer with Mg10x Standard Buffer without Mg25 mM MgCl

2

dNTPs mix

1x600 U EPTQKC610 EPTQKC620

1x2000 U EPTQKC612 EPTQKC622

12x600 U EPTQK600

Taq DNA Polymerase 5 U/µl10x Standard Buffer with MgdNTPs mix

1x250 U EPTQKD101 EPTQKD251



Taq DNA Polymerase 5 U/µl10x XD Buffer with MgdNTPs mix

1x250 U EPTQKX101 EPTQKX251



Taq DNA Polymerase 5 U/µl10x (NH

4)2SO4 Buffer without Mg50 mM MgCl2 dNTPs mix

1x250 U EPTQS001K

10x250 U EPTQS901K

Taq DNA Polymerase 5 U/µl5x Direct Loading Buffer Buffer with Mg5x Direct Loading Buffer Buffer without Mg

dNTP mix 25 mM MgCl2

1x250 U EPTQKL101 EPTQKL251

5x250 U EPTQKL105 EPTQKL255

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Taq DNA polymerase with Direct Loading Buffer> Fast and convenient

> Higher performance

> Direct loading onto gels

> Decreases cross-contamination risks

> Particularly useful in high throughput experiments

Analysis of PCR results has never been so fast and convenient

A Direct Loading buffer is provided with Taq DNA polymerase and serves as both reaction buffer and gel-loading solution. The buffer contains a densifying agent so that samples sink easily into the wells and a red/purple dye that allows direct monitoring on agarose gels.Use of “Direct loading” Products avoids repetitive pipetting steps and reduces the risk of contamination.

Highly sensitive PCRDetection of as little as 1fg of DNA has been achieved.

High yield PCRAmplification equal and in some cases superior to Taq DNA polymerase with conventional buffer.

Cat No. Product Size

EPTQL025 Taq DNA Polymerase with Direct Loading 250U

EPTQL100 Taq DNA Polymerase with Direct Loading 1000U

EPTQKL105 Taq CORE Kit 10 –Direct Loading 5 x 250U + dNTPs Mix at 10mM each

EPTQKL255 Taq CORE Kit 25 –Direct Loading 5 x 250U + dNTPs Mix at 25mM each

EPTAL100 Taq-&LOAD Mastermix 1ml (100 rxn)

EPTAL110 Taq-&LOAD Mastermix 10 x 1ml (1000 rxn)

Fig. 1 :Amplification of a 400bp fragment from a pBR plasmid with Taq-&LOAD.Reaction conditions : 5µl of Taq-&LOAD Mastermix in a final 25µl reaction. 10µl of the reaction have been loaded on a 1.2% TBE gel.

JULES

50 10 1 0.1pg 50 10 5 1fg

400 bp

MWM Taq + DirectLoading Buffer Competitor P

Taq + conven-tional buffer

1U 2U 1U 2U

7 kb

Fig. 2 :Amplification of a 7kb fragment from l DNA. Reaction conditions : 50µl reaction ; 10 ng l DNA, 200µM each dNTP, 50 pmol each specific primer, 1 or 2U of Taq DNA polymerase.10µl of the reaction have been loaded on a 1% TBE gel.

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Multiplex PCR: key factors for success

Multiplex PCR is a variant of PCR which enabling simultaneous amplification of many targets of interest in one reaction by using more than one pair of primers. Since its introduction, this method has been applied in many areas of DNA testing, including analyses of deletions, mutations, and polymorphisms. Typically, it is used for genotyping applications where simultaneous analysis of multiple markers is required, detection of pathogens or genetically modified organisms (GMOs), or for microsa-tellite analyses. Multiplex assays can be tedious and time-consuming to establish, requiring lengthy optimization procedures.Early studies highlighted the obstacles that can jeopardize the production of sensitive and specific multiplex assays, but more recent studies have provided systematic step-by-step protocols and technical improvements for test design. The most useful of these are the empirical choice of oligonucleotide primers and the use of hot start-based PCR methodology. The presence of more than one primer pair in the multiplex PCR increases the chance of obtaining spurious amplification products, primarily because of the formation of primer dimers. These non specific products may be amplified more efficiently than the desired target, consuming reaction components and producing impaired rates of annealing and extension. Thus, the optimization of multiplex PCR should aim to minimize or reduce such non-specific interactions. A straightforward solution to these difficulties has been the use of hot start PCR which often eliminates non-specific reactions (particularly production of primer dimers) caused by primer annealing at low temperature (4 to 25°C) before the beginning of thermocycling.

SurePRIME™ DNA polymeraseIt is a highly purified form of recombinant Taq DNA polymerase that has been chemically modified. In this inactivate state, the polymerase is incapable of extending primer-dimers or mis-annealed primer-template associations prior the activation step at 95°C.

> Easy-to-use: set up your 96 well plates or mastermixes at room temperature

> Low background: introduce restriction site or mismatched bases primers

> Highly specific DNA amplification: multiplex or short primers application

PCR assays were carried out using a non-optimised primer pair, amplifying a well-characterised 400 bp fragment on a human ß-globin gene. Primers are shortened oligos, with additional non-matched bases at the 5’ end, to create a restriction site at each extremity of the PCR fragment. SurePRIME™ DNA polymerase (lane 1: 1 U; lane 2: 2U; lane 3: 5 U) and classical Taq DNA Pol (lane 4: 1 U; lane 5: 2 U; lane 6: 5 U) were comparatively tested.

SurePRIME™ Taq DNA Polymerase

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Q-Bio Taq DNA polymerase

> Recombinant, truncated form of Thermus aquaticus

> Highly thermostable (98°C)

> No 5’-3’ exonuclease activity

> Enzyme cannot degrade the 5’ end of primers: specially recommended for Multiplex and RAPDs

Q-BioTaq DNA Polymerase is a thermostable DNA polymerase which can be used effectively for PCR and DNA sequencing by the chain termination method. This enzyme is encoded by a modified form of the Thermus aquaticus DNA polymerase gene with a N-terminal deletion. The properties of Q-BioTaq DNA polymerase include high thermostability and absence of 5’-3’ exonuclease activity. The enzyme is highly purified and free of RNases and endo- and exonucleases. The absence of 5’-3’ exonuclease activity and high thermostability of this enzyme make it particularly useful in applications where multiple amplifications are required.

Cat No. Product Size

EPQBT010 Q BioTaq 5U/µl 100U




EPQBT015 Q BioTaq 5U/µl 5 x 100U

EPHSP025 SurePRIME™ 5U/µl 250U

EPHSP525 SurePRIME™ 5U/µl 5 x 250U

How to avoid point mutation in PCR?Even when using Proofreading DNA polymerase, the amplification of 2.5 kb fragments could result in product with isolated point mutation.Some tips to overcome mutations:

1. Increase the template quantity 2. Decrease the cycles number3. Reduce dNTPs concentration to 100µM or 40µM4. Use the polymerase with the lowest error rate of the market, like Isis™ DNA polymerase.

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ISIS™

ISIS™ High Fidelity DNA Polymerase

High fidelity is a key requirement for all kinds of PCR applications and in particular for cloning lengthy and difficult DNA strands. Isis High Fidelity DNA polymerase is able to amplify long DNA fragments with one of the lower error rates of the market. Moreover, this polymerase ex-hibits very high thermal stability and then the denaturation temperature can be increased for reading through difficult secondary structure.

40 X more accurate than Taq DNA Polymerase

Isis DNA Polymerase is a high performance proofreading DNA polymerase. This enzyme has extremely low error rate and is 40 times more accurate than Taq DNA Pol, according to the method of Flaman (1,2). In other terms, it means that the probability of misincorporation is around one error per 1 600 000 base pairs, per PCR cycle. Isolated from Pyrococcus abyssi (3,4) this polymerase was developed in collaboration with IFREMER (Institut Français de Recherche pour l’Exploitation de la Mer).

Highly Thermostable : GC rich fragments applicationIsis DNA Polymerase has a very high thermostability and therefore, exceptional resistance to thermo-cycling compared to other proofreading DNA polymerases. This feature leads to use high temperature cycling when GC rich region will be amplified, without compromising product yield. Isis retains more than 50% polymerase activity after 5 hours incubation at 100°C.

(1) Flaman, J.M. et al. (1994) Nucleic Acids Research 22 (15) 3259-3260(2) Flaman, J.M. et al. (1995) Proc. Nat. Acad. Sci. USA 92, 3963-3967(3) J. Dietrich et al. (2002) FEMS Microbiology Letters 217; 89-94.(4) Gueguen et al (2001) Eur.J. Biochem. 268, 1-10.

Exclusive

MP Biomedicals

Product

General Applications• Cloning• Mutation Analysis• Long and accurate PCR from GC rich templates• Site-directed mutagenesis

Polymerase Cat. # Pack size Pack Included

Isis™

High Fidelity

EPSIS100 100 U 1U/µl 10x Incubation Buffer with MgSO4

30 mM MgSO4EPSIS103 3x100 U 1U/µl

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Figure 1: PCR amplification of exogenous genomic human DNA (10 ng) in presence of fresh human blood collected with EDTA (% v/v). A: 0.5 U Isis DNA Pol per 50 µl assay. Blood quantities in %v/v: 10%; 5%; 2%; 0.2% and without blood. B: 1.25 U Taq DNA Pol per 50 µl assay. Blood quantities in % v/v: 1%; 0.2%; 0.02% and without blood. M1= 420 bp fragment marker.

Figure 2: Blood direct amplification of 420 bp beta-globin fragment. Fresh human blood collected with EDTA as anti-coagulant and stored at +4°C. No external DNA is added. A: 0.5 U of Isis DNA Pol per 50 µl assay. Blood quantities in %v/v: 10%; 5%; 2%; 0.2% and without blood. Molecular weight marker (M2) is pBR322 Hae III digest. B: 1.25 U of Taq DNA Pol per 50 µl assay. Various concentrations of blood were tested: 1%; 0.2%; 0.02% and without blood. The marker (M1) is a 420 bp band.

Traditional testing methods in clinical and research laboratories are increasingly being replaced by Molecular Diagnostic tech-niques where the most popular, powerful and useful is PCR. The reasons to this change are the gain in sensitivity, specificity and rapidity in testing time.The presence of a variety of endogenous and exogenous DNA polymerase inhibitors in blood samples, make necessary to have a highly purified template to amplify.Initial sample preparation prior to the PCR setup is still a major bot-tleneck in routine sample testing which need a high degree of stan-dardisation with low labour intensi-

Isis™ DNA polymerase overcomes PCR inhibitors contained in Whole blood samples.

A B

M1 10 5 2 0.2 0 % M1 1 0.2 0.02 0 %

10 5 2 0.2 0 M2 1 0.2 0.02 0 M1

A B

Technical Note

ty and reasonable cost. Moreover, DNA isolation procedures increase the risk in cross contamination between samples.MP Biomedicals brings a solution to overcome this time consuming and problematic step by direct amplification of template from whole blood samples.Proofreading DNA polymerase Isis have shown particularly higher resistance to inhibitors compared to popular Taq DNA polymerase at lower Unit quantity and with the benefit of 40x times more accurate results. The high fidelity property is of high interest when used in SNP tests.Figure 1 compares Isis and Taq DNA Polymerase performances to

detect Beta-globin gene from 10ng exogenous gDNA, in presence of different volumes of blood with EDTA as anticoagulant. Assays using up to 5% (vol/vol) are effi-ciently performed with 0,5 U of Isis while 1,25U Taq DNA is only able to perform up to 0.2% of blood. Isis DNA Pol exhibits an over perfor-mance of 25x versus Taq DNA Pol.Identical PCR assays were performed without addition of external DNA. Isis DNA Pol is able to amplify the target in presence of 0.2% to 5% blood, whereas Taq DNA Pol is ineffective (Fig 2). These results were also confirmed with sodium citrate as anticoagulant.

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MP Biomedicals : Your partner at every stage of your research project

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Sample lysis PCRPurification Further applications

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PCR Brochure 2010