Biochem 121.1 Biochemistry of the Gene Laboratory

Bacteria and Blood DNA Extraction

Lopez, Richard; Palmario, Marijyke Katrina Patricia; Tan, ElaineGroup 2, Section MCDMarch 14, 2011Professor Joanne del Rosario

I. AbstractGenomic DNA extraction from bacteria and whole blood was performed in

this experiment. For bacterial genomic DNA extraction, the method adapted from Tillett and Neilan was used. Mainly, the method was composed of obtaining the lysate, salting out and selective precipitation. For genomic DNA extraction from whole blood, the Wang’s Lysis Method was used. The method consisted of lysis to remove the erythrocytes which did not contain nuclei, salting out and selective precipitation, as well. Both methods are rapid, efficient, safe and can serve as standard laboratory protocols. Analysis using gel electrophoresis in 1x TAE viewed under UV light is also presented in the paper.

II. Keywords

DNA Extraction, Bacterial Genomic DNA, Blood Genomic DNA, Tillett and Neilan Method, Wang’s Lysis Method

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III. Introduction

The total genetic information of an organism is specified by its genomic DNA. In almost all organisms, including prokaryotes and eukaryotes, it is the DNA which comprises the genome. Viruses are the only exception to this fact because they have RNA genomes.

Since it constitutes the total genetic information, genomic DNA is generally large. In addition to this, complexes of DNA-protein are organized in chromosomes. These characteristics, such as the size of the genomic DNA, its nature, and the number of chromosomes vary from organism to organism.

As for prokaryotes, specifically bacteria, chromosome is single in number and circular in shape. On the other hand, as for eukaryotic organisms, chromosome is linear in shape and is multiple in number.

Aside from chromosomes, one can also consider the number of base pairs of an organism as distinct characteristics. For example, the Lambda phage has 48, 502 base pairs. Man, Homo sapiens, has approximately 3.3 x 109 and corn, Zea mays, has 3.9 x 109 base pairs. The molecular weight of the genome, measured in Daltons, is also important in DNA analyses.

Today, DNA of organisms is usually extracted because it is an invaluable tool in the fields of

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Molecular Biology, Biochemistry and Forensic Analysis. However, the process does not start at extraction or isolation alone. It actually starts on sample storage before the isolation of genomic DNA. Indeed, this is the most crucial step since it is the quality of the starting material which will affect the quality and yield of the isolated DNA.

For bacteria, sample storage is relatively simple. Cold storage, at a temperature range of -20 0C to - 80 0C, is sufficient. However, DNA extraction from animal tissues, especially blood, poses several challenges.

In blood DNA extraction, a problem encountered is that mammalian erythrocytes or red blood cells do not contain nuclei. Consequently, genomic DNA cannot be extracted from RBC and these must therefore be eliminated. Upon elimination of erythrocytes which comprise majority of the blood sample, the leukocytes which contain nuclei remain. Hence, genomic DNA can be extracted from blood.

For the isolation proper, DNA can be separated from cellular components using four main steps, namely disruption, lysis, removal of proteins and contaminants, and then recovery of DNA.

In general, there are several methods available for the extraction of DNA. However, regardless of the methods used, the five main steps which include storage, disruption, lysis, removal of contaminants and recovery of DNA still hold true in separating DNA from cellular components.

In laboratories, there are seven common protocols used for DNA extraction. Each method has its advantages and disadvantages, as will be discussed here.

The first method is termed as the preparation of crude lysates. Here, the cell lysate is simply incubated at high temperatures or digested with proteinase K. The recovered product will then be an impure and contaminated genomic DNA. This technique is not advisable since it only gives rise to high failure rates.

Another technique is the salting-out method. This method is actually a refinement of the crude lysate. Here, the proteins and contaminants are precipitated from the cell lysate using high salt concentrations. Ammonium acetate and potassium acetate are usually used as salts. However, DNA yield and purity are not definitive in this technique.

Organic extraction methods are also available for DNA extraction. Here, lysis is performed by the use of a detergent, Afterwards, the lysate is mixed with isoamyl alcohol, polar and non-polar reagents. The polar reagent is usually phenol and the non-polar reagent is usually chloroform. Upon mixing, the components are then separated into two phases – organic and aqueous. Contaminants are in the organic phase and DNA molecules are in the aqueous phase. However, both phenol and chloroform may leave residues in the DNA sample. More importantly, these reagents pose health hazards.

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The principle behind that of the organic extraction method is the same as that of the CTAB method which was not performed in class due to the aforementioned disadvantages.

In the Cesium Chloride Density Gradient Method, the cell lysate is first precipitated using alcohol. Upon doing so, resuspended DNA is mixed with both CsCl and EtBr, then centrifuged. The DNA band is then collected and then washed with alcohol to remove EtBr. Not only is this method tedious and expensive, it also requires EtBr which is a known mutagen.

The sixth method is the anion-exchange method. In this method, the phosphates of the nucleic acids which have a negative charge adhere to the positive charged surface molecules on the substrate. Using medium-salt washing, the impurities are washed out and DNA can be eluted using a high-salt buffer. The problem with this method is its high cost.

Lastly, silica-based methods such as kits can be utilized. Here, nucleic acids selectively adsorb on the silica-gel membrane, in the presence of high concentrations of salts. Several buffers also ensure that contaminants do not adsorb on the membrane. DNA can then be finally eluted out of the membrane. As easy as it may seem, this method is expensive to use in undergraduate laboratory courses.

The methods discussed above are some of the ones used in genomic DNA extraction from bacteria.

In animal tissues, such as blood, the Wang’s Method can be used due to its several advantages.

The Wang’s Method was first published in 1994. It was established in order to have a standard protocol for rapid and efficient DNA analysis. At that time, extraction of DNA either heavily relied on toxic reagents such as phenol and chloroform, or on tedious manipulations such as literally spooling DNA.

The Wang’s Method is characterized not only by its rapidity and efficiency but also by its reproducibility, high yield and high purity.

Figure 1. The figure shows the reproducibility of the method with different sample volumes (Wang, et. al., 1994).

In the fields of Biochemistry and Molecular Biology, DNA recovered from the said method can be used for electrophoresis since a high molecular weight is preserved, for restriction endonuclease digestion and for PCR template.

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Figure 2. Electrophoresis of Purified Genomic DNA from Human Whole Blood by Wang’s Method and of its Digests from Restriction Endonucleases (Wang, et. al., 1994).

As can be seen in Figure 2, Lane 1 serves as the marker for HindIII digest. Lane 2 contains the recovered genomic DNA which shows desirable molecular weight. Lanes 3 to 5 contain its digests from BamHI, EcoRI and HindIII. The figure supports the claim that Wang’s method can indeed be used for several applications involving purified DNA.

In this paper, the extraction of genomic DNA from bacteria and animal tissue, specifically blood, will be discussed. Also, in this paper, the methodology used in bacterial DNA extraction is the Xanthogenate-SDS Method (Tillet and Neilan, 2000). In blood DNA extraction, the Wang’s Lysis Method (Lu Wang, et. al., 1994) is used. Upon discussion of the extraction procedure, results and qualitative analysis of the recovered DNA will be presented.

IV. Methodology

The method used for DNA extraction from bacteria is different from that of the DNA extraction from animal tissues, specifically blood. The two methods are described below.

A. DNA Extraction from Bacteria

The procedure for DNA extraction from bacteria, called Xanthogenate-SDS Method, is adapted from Tillet and Neilan, 2000. In this method, the TER buffer, XS buffer, isopropyl alcohol, 70% ethanol and TE buffer were the primary reagents used.

The TER buffer is composed of 10 mM Tris-HCl at pH 7.4, 1 mM EDTA at pH 8.0 and 100 ug/mL RNAse A.

The XS buffer is composed of 1% potassium ethyl xanthogenate, 100 mM Tris-HCl at pH 7.4, 1% Sodium Dodecyl Sulfate and 800 mM ammonium acetate.

Lastly, the TE buffer was maintained at a pH of 8.0.

As for the procedure, 10-20 mg sample was first placed in a 1.5 mL microcentrifuge tube. Afterwards, it was re-suspended in 50 uL TER buffer. 750 uL of freshly prepared XS buffer was then added to the tube. Inversion was done several times to mix the components.

For 60 minutes and at 70 0C, the microcentrifuge tubes were incubated in a waterbath. After doing so, the tubes were vortex-mixed for 10 seconds. Next, the tubes were placed on ice for 30 minutes.

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To precipitate all cell debris, the tubes were centrifuged at 14,000 rpm for 10 minutes. The supernatant obtained was then transferred to a new microcentrifuge tube containing isopropyl alcohol. Incubation at room temperature was done for 10 minutes.

After incubation, the samples were centrifuged at 12,000 rpm for 10 minutes. Finally, the DNA pellets were washed with 70% ethanol and then were allowed to air dry. The pellet was then re-suspended in 100 uL TE buffer.

B. DNA Extraction from Blood

The procedure for DNA extraction from blood used the Wang’s Method. But first, a whole blood sample was first extracted.

The main reagent used for this procedure is the Wang’s Lysis solution which is composed of 1% w/v Triton X-100, 0.32 M Sucrose, 5mM MgCl2 and 10 mM Tris-Cl at pH 7.5.

There are other reagents aside from the Wang’s Lysis Solution. These reagents include the enzyme reaction solution, sodium iodide solution, Tris-EDTA buffer, 70% ethanol, 40% absolute isopropanol and 20 mg/mL Proteinase K stock solution stored at -20 0C.

The enzyme reaction solution is composed of 1% w/v SDS, 5 mM EDTA and 10 mM Tris-Cl at pH 8.0.

The sodium iodide solution, stored in the dark at room temperature, is composed of 7.6 M

NaI, 20 mM EDTA and 40 mM Tris-Cl at pH 8.0.

Lastly, the TE or Tris-EDTA buffer was composed of 10 mM Tris at pH 8.0 and 1 mM EDTA.

As for the procedure, approximately 8 mL of blood was first extracted. To the sample, 1 mg/mL EDTA-Na2 was added. The blood solution was then divided into 0.5 mL aliquots in microcentrifuge tubes.

Wang’s lysis solution, in an amount of 0.5 mL, was added next. The resulting solution was then centrifuged at 10, 000 x g for 20 seconds.

The supernatant was discarded, 1 mL of lysis solution was added to the pellet and then the resulting solution was mixed by inversion for 30 seconds. Centrifugation at 10, 000 x g for another 20 seconds was performed. The addition of 0.5 mL Wang’s lysis solution and centrifugation was also repeated.

After centrifugation, the pellet was re-suspended in 0.2 mL of enzyme reaction solution. Incubation at 370C for 10 minutes was performed next. Upon doing so, 10 uL of Proteinase K solution was added and incubated at 1 hour for 37 0C, with occasional mixing. 0.3 mL of NaI solution was added, and then gently inverted to mix. 0.5 mL of isopropanol was also added and then gently inverted.

Centrifugation was once again performed, at a rate of 10, 000 x g for 10 minutes. Decantation was used to remove the supernatant.

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The pellet remained and to it, 1 mL of 40% isopropanol was added. Centrifugation at 10, 000 x g was performed for 5 minutes. The supernatant was also removed.

The above step was performed once again, but 70% ethanol was used instead of 40% isopropanol.

Finally, the resulting pellet was air-dried for 10 minutes and then re-suspended in 0.1 mL TE buffer, maintained at pH 8.0.

V. Results and Discussion

Out of these seven methods discussed in the Introduction, the method used for the DNA extraction from bacteria is a combination of the first and second. To be more specific, it is the Xanthogenate-SDS Method by Tillett and Neilan, 2000.

DNA extraction starts with the proper storage of the DNA sample. Afterwards, disruption and lysis begins.

Disruption and lysis can be performed using a TER buffer. A TER buffer is composed of Tris-HCl, EDTA and RNAse A. Each component has its function.

EDTA,ethylenetriamine tetraacetic acid, is a known chelating agent. It binds divalent cations, specifically Magnesium and Calcium which maintain the cell membrane’s integrity. Upon binding with the cations, the membrane is disrupted and lysed.

Tris(hydroxymethyl) aminomethane, or Tris, works as a biological buffer. A buffer is of tremendous importance in this

experiment because DNA is highly pH-sensitive. The pKa of Tris is equal to 8.1, which means that it can prevent drastic pH shifts ad maintain the pH at a range of 7.0 to 9.0. The second role of Tris is to interact with the lipopolysaccharide membrane and make it more permeable in order for disruption and then lysis to occur.

Since the membrane has been disrupted and lysed, debris such as RNA must be removed. RNAse A is used to digest contaminating RNA.

However, bacterial cells have rigid cell walls that are rich in polysaccharides. Consequently, these are difficult to rupture completely. Enzymes have been used to rupture these cell walls but the cells are not thoroughly lysed and DNA yield is poor. Thus, the XS buffer is added next.

The XS buffer is composed of potassium ethyl xanthogenate, Tris-HCl, sodium dodecyl sulphate and ammonium acetate.

Potassium ethyl xanthogenate is the salt of a xanthic acid. It is formed by the reaction of potassium hydroxide, ethanol and carbon disulfide. In this experiment, its main role is to dissolve the cell wall of bacteria. Upon dissolution, water-soluble polysaccharide xanthates are formed. Moreover, these xanthate-forming compounds can inhibit DNAse activity, by binding to Magnesium and Calcium divalent cations. Indeed, it serves to rupture the cell wall and to destabilize the membrane.

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Another component of the XS buffer is SDS or Sodium Dodecyl Sulfate. Its main function is to act as anionic surfactant and denature the proteins that may act as contaminants in the DNA sample. Denaturation occurs because it is amphiphillic and disrupts the non-covalent bonds of proteins.

Ammonium acetate is also a component of the XS buffer. It is the salt of acetic acid and ammonia. Approximately, it has a pKa of 7.0. A salt, in this experiment, functions to precipitate the proteins out of the solution.

After the TER and XS buffer have been added, the tubes were incubated at 70 0C for an hour. Incubation was done to ensure that complete reaction took place.

At this point, it can be inferred that RNA, protein and other possible contaminants have been removed from the “cellular soup”. At the same time, the buffer served to maintain the pH of the solution at the physiological level.

To further separate the debris from DNA, vortexing the DNA at 10 seconds was done. Care was observed in vortexing since genomic DNA is large and too much agitation can shear the DNA. In addition, the solution was subject to an ice bath to aid in the precipitation and then centrifuged to finally separate the other cellular components from DNA.

Since the genomic DNA is heavier, it remained in the supernatant. Afterwards, it was treated to isopropanol. This was done to precipitate the DNA, since DNA is insoluble in polar

substances, such as alcohol. Aggregation then occurs, and then a white pellet is finally formed.

To remove excess alcohol and those salts which are soluble in alcohol, centrifugation was once again performed. Washing in 70% ethanol was done next, since ethanol is volatile and easily volatilizes itself from the DNA.

Finally, the pellet was resuspended in TE buffer. A buffer is needed, and not water, since DNA hydrolyzes in water; whereas, its pH is controlled in the presence of a buffer.

The above mentioned protocol is for bacterial DNA isolation. Some of its steps are similar to that of DNA isolation from human tissues, specifically blood; whereas, some are not. In this paper, the method used for DNA extraction from blood is that of Wang’s.

DNA isolation from blood starts with the proper storage of the sample. For optimum conditions, it must be stored at a temperature of -20 0C to - 80 0C.

The first step, upon obtaining the stored sample, is the addition of an anticoagulant. This is needed so as to avoid blood clotting and be able to obtain whole blood. In the market, the three main anticoagulants used are EDTA-Disodium, Heparin and Citrate. Citrate gives poor DNA yields. The mode of action of heparin consists of inhibiting thrombin. Consequently, upon inhibition, fibrinogen is not converted to fibrin and blood does not clot. However, the problem concerning heparin is

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that it interferes with downstream applications of recovered DNA (Beautler, et. al., 1990), though the exact mechanism by which it interferes has not been elucidated yet.

For this reason, EDTA-Disodium was chosen to be the anticoagulant for this experiment. The said reagent works by chelating the calcium ions, which render these inactive to participate in coagulation. Moreover, EDTA-Disodium does not interfere much with recovered DNA downstream applications.

A study (Ty, et. al, 2008) shows that there is no significant difference between the yield of recovered DNA using EDTA-Disodium and Heparin. Indeed, choosing EDTA-Disodium over Heparin is a matter of less interference in downstream PCR applications by the former reagent.

Figure 3. Average DNA yield using three common anticoagulants: EDTA, Citrate and Heparin, respectively. (Ty, et. al., 2008)

Upon addition of anticoagulant, the blood was divided into aliquots and Wang’s Lysis Solution was added.

Wang’s Lysis Solution has several components. These are

Triton X-100, sucrose, Magnesium Chloride and Tris-Cl.

Before discussing the components of the lysis solution, it is important to note that mammalian red blood cells or erythrocytes do not contain nuclei. Hence, genomic DNA cannot be extracted from these. Another fact is that in whole blood, there are approximately 1000x more erythrocytes than leukocytes which contain nuclei. Thus, eliminating erythrocytes is needed in order to recover DNA that is high in both yield and purity.

The elimination of erythrocytes was done using the Wang’s Lysis Solution. Triton X-100, scientifically known as octylphenolpoly(ethyleneglycolether)x, is a component of the solution and it functions as a non-ionic detergent. Mainly, it solubilises the proteins of the cell membranes, rendering these easier to disrupt.

Sucrose is another component of the lysis solution. It selectively lyses erythrocytes by hypotonic shock, since these are more susceptible than leukocytes.

The next component is Magnesium Chloride. It is a salt and it functions to selectively precipitate nuclei-containing cells out of the solution. Also, the Magnesium cations counteract the Phosphate anions of the nucleic acids and protects DNA from degradation. In addition to this, the buffer protects the solution from pH shifts since the cell debris generated can drastically alter it.

It can also be remarked that the volume of the blood sample must be equal to that of the lysis solution

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so as to ensure complete lysis of the erythrocytes.

Upon lysis, repeated centrifugation was performed in order to separate the debris from the cellular pellet.

When the supernatant was discarded and the pellet was obtained, the enzyme reaction solution was added. The enzyme reaction solution contained SDS, EDTA and Tris-Cl.

As discussed earlier, SDS works as an anionic detergent that disrupts the cell membrane, EDTA chelates divalent cations and disrupts cell integrity and Tris-Cl works as a buffer. Incubation was done so as to ensure complete lysis of the cell.

Upon incubation, Proteinase K was added. It is a protease with broad specificity, has a high activity and can digest proteins at short times. Its optimum temperature range is from 65 0C to 70 0C. Hence, the sample was incubated at 70 0C to ensure complete protein digestion.

A distinct characteristic of Proteinase K that makes it suitable for use is that it does not need divalent cations, such as Magnesium and Calcium. Hence, EDTA can chelate these ions and Proteinase K can still perform its work of digesting proteins.

Since the proteins have already been digested by the protease, sodium iodide was added next. Sodium iodide is a crystalline salt that must be stored in the dark at room temperature because it can readily decompose. Furthermore,

low temperature can cause it to recrystallize.

Biological compounds such as polypeptides and proteins remain soluble in high concentrations of NaI; whereas, DNA are insoluble. Thus, further addition of isopropanol was able to selectively precipitate DNA out of the solution. Consequently, a white-pellet was formed.

Centrifugation was once again performed to separate the pellet from the supernatant. The pellet was treated with 40% isopropanol to further remove alcohol-soluble proteins and ensure that high purity DNA is recovered.

Now that the DNA pellet has been purified, 70% ethanol was added to it. Air-drying was performed next. Ethanol easily volatilizes; thus, what remains in the solution is DNA.

Lastly, DNA was re-suspended in TE buffer. TE buffer, not water, was used since DNA hydrolyzes in water, whereas, the buffer protects it from pH shifts.

The recovered DNA can be used for several purposes. It can be applied in PCR, restriction enzyme digestion, molecular cloning and DNA sequencing.

After agarose gel electrophoresis in 1x TAE was performed on the recovered DNA, it was viewed under UV light. The resulting electrophoresis is as follows:

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Figure 4. Electrophoresis of Genomic DNA Purified from Bacteria (Lanes 1-4) and Blood (Lanes 7-9). Lanes 5, 6 and 10 served as markers.

In Lane 1, no distinct band can be seen. Mishandling of the DNA might have occurred in the process of disposing the supernatant. The pellet, which might have been too small to settle at the bottom of the microcentrifuge tube, might have been disposed of. Thus, supernatants must be kept until the analysis of DNA has been completed since it might contain valuable DNA.

In Lanes 2 to 4, the distinct bands show the high-molecular weight of bacterial genomic DNA. The faded bands at the bottom show cellular debris, particularly RNA, which was not degraded by RNAse A since the enzyme was not available in the laboratory.

Lanes 5 and 6 contained the blood marker. No observable band was seen; hence, no DNA was extracted. The cause of this problem can be attributed to the storage of DNA. Earlier, it was discussed that blood samples for DNA isolation must be kept at a

temperature range of -20 0C to - 80 0C. Such range was not available and DNA became destabilized.

Lanes 7 to 9 should theoretically contain blood from genomic DNA. Such was not the case. Several mistakes can cause this. The blood sample might have not been properly stored, the lysis solution might have not been enough to lyse the erythrocytes or the small pellet might have been disposed of.

Lastly, Lane 10 served as the marker for the experiment.

Another figure shows the analysis using electrophoresis, this time using a kit for bacterial genomic DNA extraction.

Figure 5. Electrophoresis of Purified Genomic DNA of Bacteria from kit (Lanes 1 and 2), Bacteria from Standard Protocol (Lanes 3 and 4) and blood (Lanes 7 and 8). Lanes 5, 6 and 9.

As can be seen in this figure, using DNA kits does not ensure a no-fail experiment. No DNA bands can be observed from that of Lanes 1 and 2 since the water bath was not maintained at 70 0C and lysis might have not occurred. Indeed, only proper handling of the

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experiment protocol can ensue that DNA is recovered.

VI. Conclusion and Recommendations

At the end of the experiment, it can be concluded that bacterial DNA was successfully extracted using the Tillett and Neilan Method.

This is supported by the fact that distinct bands were seen in the gel electrophoresis.

Indeed, the protocol used was efficient, fast, relatively cheap and safe, when compared to organic extraction methods.

A kit was also employed in this experiment. However, a kit does not always guarantee a better result than manual techniques since the proper handling of the samples and reagents is still the key to a successful experiment.

For genomic DNA extraction from whole blood, the Wang’s Lysis Method was used.

As can be seen in the gel electrophoresis, no bands were observed due to both the improper storage of the samples and practice of laboratory techniques.

The recovered DNA from these experiments has several applications. These include PCR, Southern Blotting, molecular cloning and DNA sequencing, to name a few.

VII. References

1. Tillett, D. and B. A. Neillan. Xanthogenate Acid Isolation from Cultured and Environmental Cyanobacteria. Journal of Phycology, 2000.

2. Wang, et. al., Purification of Genomic DNA from Human Whole Blood by Isopropanol-Fractionation with Concentrated NaI and SDS. Japan: Osaka Research Laboratories, 1994.

3. Khosravinia, et. al. Influence of EDTA and Magnesium on DNA Extraction from Blood Samples and Specificity of Polymerase Chain Reaction. India: Lorestan University, 2006.

4. Ty, et. al., Isolation of DNA from Blood Samples and Body Fluids Using E-Z 96 Mag-Bind Blood System. VWR International, Issue 21, 2008.

5. Beutler, et. al. Interference of Heparin with the Polymerase Chain Reaction. Journal of Biotechnology, 1990.

I hereby certify that I have given substantial contribution to this

report.

Lopez, Richard

Palmario, Katrina Marijyke

Tan, Elaine

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