Random-Primed, Phi29 DNA Polymerase-Based Whole Genome Amplification
UNIT 15.13
John R. Nelson1 1
GE Global Research, Niskayuna, New York
ABSTRACT Whole-genome amplification by multiple displacement amplification (MDA) is a patented method to generate potentially unlimited genomic material when researchers are challenged with trace samples, or the amount of genomic DNA required for analysis exceeds the amount on hand. It is an isothermal reaction, using Phi29 DNA polymerase and random hexamer primers for unbiased amplification of linear DNA molecules, such as genomic DNA. The random-primed MDA reaction provides extensive amplification coverage of the genome, generates extremely long DNA products, and provides high DNA yields. This unit explains the reaction, and describes use of the commercial kits C 2014 by John Wiley & Sons, available. Curr. Protoc. Mol. Biol. 105:15.13.1-15.13.16. Inc. Keywords: multiple displacement amplification (MDA) r whole-genome amplification (WGA) r Phi29 DNA polymerase r isothermal amplification
INTRODUCTION The whole-genome DNA amplification method takes advantage of the fact that Phi29 DNA polymerase has excellent strand-displacement activity when replicating DNA (Blanco et al., 1989). Using random hexamer primers to initiate amplification of genomic DNA samples, this single subunit, proofreading DNA polymerase will initiate synthesis at many random loci and, as the template strand is replicated, any doublestranded sections of DNA encountered by the enzyme are displaced off the template. These displaced strands of single-stranded DNA are also randomly bound by primer and replicated in a cascading reaction that typically continues until nucleotides in the reaction mixture are depleted (Fig. 15.13.1). Simple phosphorothioate modification of the random sequence primers prevents the DNA polymerase from degrading the primers and dramatically stimulates reaction kinetics (Dean et al., 2001). The reaction has been shown to provide as much as 106 -fold amplification (Kumar et al., 2008). Large amounts of amplified product DNA can be obtained in just a few hours. Additionally, the presence of an associated proofreading function within the Phi29 DNA polymerase ensures high-fidelity amplification. The error rate of this method has been measured at one in 3 × 105 bases (Nelson et al., 2002). The product DNA can be used directly in most DNA analysis methods as a direct replacement for genomic DNA. In this unit, methods for performing whole-genome amplification using the commercially available kits from both GE (see Basic Protocol 1 and Alternate Protocol 1) and Qiagen (see Basic Protocols 2 and 3) are explained. Basic Protocols 1 and 2 describe utilization of the GenomiPhi DNA Amplification kit and the REPLI-g Mini Kit, respectively, for amplification of purified genomic DNA. Alternate Protocols 1 and 2 describe utilization of the GenomiPhi DNA Amplification kit and the REPLI-g Mini kit, respectively, for amplification of genomic DNA from trace cellular material. The Support Protocol describes The Polymerase Chain Reaction Current Protocols in Molecular Biology 15.13.1-15.13.16, January 2014 Published online January 2014 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/0471142727.mb1513s105 C 2014 John Wiley & Sons, Inc. Copyright
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genomic DNA random primer
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Figure 15.13.1 Random hexamer primers anneal to the template DNA at multiple sites. Phi29 DNA polymerase initiates replication at multiple sites on the denatured linear DNA simultaneously. As synthesis proceeds, strand displacement of complementary DNA generates new singlestranded DNA. The subsequent priming and strand displacement replication of this DNA results in the formation of double-stranded DNA.
an optional method for the determination of the precise concentration of amplified DNA product in cases where this information is needed. The Commentary section describes the limitations to the protocols and expected results. BASIC PROTOCOL 1
RANDOM-PRIMED, WHOLE-GENOME AMPLIFICATION OF GENOMIC DNA: THE GenomiPhi AMPLIFICATION KIT The purpose of this method is to amplify genomic DNA from any source using the GenomiPhi whole-genome amplification kit (GE Healthcare). It outlines the basic steps starting from purified genomic DNA, denaturing the sample, and amplifying the DNA.
Materials Sample genomic DNA illustra GenomiPhi V2 DNA amplification kit (http://www.gehealthcare.com/lifesciences) containing: Sample buffer Reaction buffer Enzyme mix Control DNA (lambda DNA) TE (see recipe) 0.5-ml plastic microcentrifuge tubes 30°, 65°, and 95°C water baths or thermal cycler 30°C incubator 1. Add 9 μl sample buffer to 1 μl of 1 to 10 ng template DNA. Template DNA should be resuspended in TE or water.
Phi29 DNA Polymerase–Based WGA
Sample genomic DNA should be as intact as possible. Fragmented genomic DNA 3 min or at higher temperatures can cause damage to the DNA.
3. Prepare the master mix. For each amplification reaction, combine 9 μl of reaction buffer with 1 μl enzyme mix on ice. Prepare the master mix only in required quantities and immediately prior to use. Keep the master mix on ice and discard any unused portion. The master mix contains all the components required for DNA amplification and will generate amplification products if exposed to temperatures >4°C for a sufficient time. Even on ice this mixture will be capable of DNA amplification, and it should be combined with sample as quickly as possible. Take precautions to maintain sterility during handling of these stock solutions to prevent contamination with extraneous DNA that could compromise product integrity. Wear gloves at all times during preparation.
4. Assemble amplification reaction by adding 10 μl of prepared master mix from step 3 to each cooled DNA sample, on ice. 5. Amplify DNA by incubating samples 1.5 hr at 30°C. During this step the phi29 DNA polymerase extends the random primers, creating new copies of the input genomic DNA while displacing upstream, newly created strands. These displaced strands are in turn primed and amplified. This cascading reaction results in exponential kinetics of amplification proceeding until the available dNTPs are depleted.
6. Inactivate the Phi29 DNA polymerase enzyme by heating samples 10 min at 65°C and then cooling to 4°C. Heating is required to inactivate the exonuclease activity of the DNA polymerase, which may otherwise begin to degrade the amplification product. The proofreading activity of phi29 DNA polymerase is active on single-stranded DNA, and will degrade single-stranded DNA in a processive manner.
7. (Optional) Analyze the product (see Support Protocol) by standard agarose gel electrophoresis to confirm that high-molecular-weight DNA has been generated. Typically, the product will migrate as a smear on the gel ranging in size from 2 to 5 kb up to >20 kb. If the precise concentration of product is required, see Support Protocol to quantify the sample.
8. Store finished amplification reactions up to 6 months at −20°C. Whole-genome DNA amplification products should be stored and treated as genomic DNA. Be sure to completely mix the sample after thawing, as the DNA can partition when freezing. For long-term storage beyond 6 months, storage at −80°C may be preferred.
WHOLE-GENOME AMPLIFICATION DIRECTLY FROM BLOOD OR CELLS: THE GenomiPhi AMPLIFICATION KIT
ALTERNATE PROTOCOL 1
The purpose of this method is to amplify genomic DNA directly from a small number of cells using the GenomiPhi whole-genome amplification kit (GE Healthcare). It outlines the basic steps starting from cells, lysing the cells while denaturing DNA, and amplifying DNA.
Materials Cell sample in PBS (see recipe for PBS) Denaturation solution (see recipe) Neutralization buffer (see recipe)
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GenomiPhi V2 DNA amplification kit (http://www.gehealthcare.com/lifesciences) containing: Sample buffer Reaction buffer Enzyme mix Control DNA (lambda DNA) 0.5-ml microcentrifuge tubes 30°, 65°, and 95°C thermal cycler or water baths 1. Lyse cells and denature genomic DNA by mixing 1 μl cells (suspended in PBS) with 1 μl denaturation solution in a 0.5-ml microcentrifuge tube. Mix by pipetting up and down two to three times. Incubate 10 min on ice. Do not vortex. Trace cellular samples in PBS can be amplified with this protocol. Samples cannot be used if they have been treated with formalin or other crosslinking reagents. While amplification from fewer than 150 cells can be attempted, the resulting amplified DNA may not be high quality. Amplification from >2000 cells may result in slower amplification kinetics or amplification failure. Blood samples should be diluted 1:3 in PBS to dilute inhibitory components. Cellular material can be washed in PBS to eliminate contaminating materials that may inhibit amplification.
2. Neutralize lysed cells/DNA solution by adding 1 μl neutralization buffer and pipetting up and down two to three times. Store on ice. Do not vortex. 3. Prepare the master mix for each amplification reaction. Combine the following for each reaction on ice:
7 μl sample buffer 9 μl reaction buffer 1 μl enzyme mix Prepare the master mix only in required quantities and immediately prior to use. Keep the master mix on ice and discard any unused portion. The master mix contains all of the components required for DNA amplification and will generate amplification products if exposed to temperatures >4°C for sufficient time. Even on ice this mixture will be capable of DNA amplification, and it should be combined with sample as quickly as possible. Take precautions to maintain sterility during handling of these stock solutions to prevent contamination with extraneous DNA that could compromise product integrity. Wear gloves at all times during preparation.
4. Transfer master mix to cooled sample by adding 17 μl of prepared master mix from step 3 to each cooled sample from step 2 on ice. 5. Incubate samples 2 hr at 30°C to amplify DNA. During this step, the phi29 DNA polymerase extends the random primers, creating new copies of the input genomic DNA while displacing upstream, newly created strands. These displaced strands are in turn primed and amplified. This cascading reaction results in exponential kinetics of amplification proceeding until the available dNTPs are depleted.
6. Inactivate Phi29 DNA polymerase enzyme by heating samples 10 min at 65°C and then cooling to 4°C.
Phi29 DNA Polymerase–Based WGA
Heating is required to inactivate the exonuclease activity of the DNA polymerase, which could otherwise begin to degrade the amplification product. The proofreading activity of phi29 DNA polymerase is active on single-stranded DNA, and will processively degrade single-stranded DNA.
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7. (Optional) Analyze product (see Support Protocol). The product of the reaction can be analyzed by standard agarose gel electrophoresis to confirm that high-molecular-weight DNA has been generated. Typically, the product will migrate as a smear on the gel ranging in size from 3 to 5 kb up to >20 kb. If the precise concentration of product is required, refer to the Support Protocol for methodology to quantify the sample.
8. Store completed amplification reactions up to 6 months at −20°C. GenomiPhi DNA V2 amplification products should be stored and treated as genomic DNA. Be sure to completely mix the sample after thawing, as the DNA can partition when freezing. For long-term storage beyond 6 months, storage at −80°C is recommended.
RANDOM-PRIMED, WHOLE-GENOME AMPLIFICATION OF GENOMIC DNA: THE REPLI-g MINI KIT
BASIC PROTOCOL 2
The purpose of this method is to amplify genomic DNA from any source using the REPLI-g mini whole-genome amplification kit (Qiagen). It outlines the basic steps starting from purified genomic DNA, denaturing the sample, and amplifying DNA.
Materials REPLI-g Mini kits (http://www.qiagen.com): Buffer DLB (reconstitute with 500 μl nuclease-free water, mix well, and centrifuge briefly) Stop solution REPLI-g mini reaction buffer REPLI-g mini DNA polymerase Nuclease-free water Template DNA 0.5-ml microcentrifuge tubes Vortex Centrifuge 30°, 65°, and 95°C thermal cycler or water baths 1. Prepare buffer D1 by mixing 0.6 μl of reconstituted buffer DLB and 2 μl nucleasefree water per sample. This is typically made as a larger cocktail for multiple reactions, and 2.5 μl of buffer D1 is used per sample.
2. Prepare buffer N1 by mixing 0.8 μl stop solution and 4.53 μl nuclease-free water per sample. This is typically made as a cocktail for multiple reactions, and 5 μl of buffer N1 is used per sample.
3. Denature template DNA by placing 2.5 μl template DNA into a 0.5-ml microcentrifuge tube. Add 2.5 μl buffer D1 and mix by vortexing. Centrifuge briefly at room temperature and incubate samples 3 min at room temperature (15° to 25°C). DNA is denatured by alkali during this step. This protocol is optimized for whole-genome amplification from >10 ng of purified genomic DNA template. The template DNA should be suspended in TE buffer. Genomic DNA should be as intact as possible. Fragmented genomic DNA 4°C for a sufficient time. Even on ice this mixture will be capable of DNA amplification, and it should be combined with sample as quickly as possible. Take precautions to maintain sterility during handling of these stock solutions to prevent contamination with extraneous DNA that could compromise product integrity. Wear gloves at all times during preparation.
6. Assemble amplification reaction by adding 40 μl master mix to 10 μl denatured DNA from step 5. 7. Incubate 10 to 16 hr at 30°C to amplify DNA. During this step the phi29 DNA polymerase extends the random primers, creating new copies of the input genomic DNA while displacing upstream, newly created strands. These displaced strands are in turn primed and amplified. This cascading reaction results in exponential kinetics of amplification proceeding until the available dNTPs are depleted.
8. Inactivate REPLI-g Mini DNA polymerase by heating samples 3 min at 65°C. Heating is required to inactivate the exonuclease activity of the DNA polymerase, which may otherwise begin to degrade the amplification product. The proofreading activity of phi29 DNA polymerase is active on single-stranded DNA, and will processively degrade single-stranded DNA.
9. (Optional) Analyze the product (see Support Protocol) by standard agarose gel electrophoresis to confirm that high-molecular-weight DNA has been generated. Typically, the product will migrate as a smear on the gel ranging in size from 2 to 5 kb up to >20 kb. If the precise concentration of product is required, see Support Protocol to quantify the sample.
10. Store amplified DNA at 4°C for short-term storage (a few weeks), or –20°C for longer-term storage (a few months). Whole-genome DNA amplification products should be stored and treated as genomic DNA. Be sure to completely mix the sample after thawing, as the DNA can partition when freezing. For long-term storage beyond 6 months, storage at −80°C is recommended. ALTERNATE PROTOCOL 2
RANDOM-PRIMED, WHOLE-GENOME AMPLIFICATION OF GENOMIC DNA FROM BLOOD OR CELLS: THE REPLI-g MINI KIT The purpose of this method is to amplify genomic DNA from trace cells using the REPLIg mini whole-genome amplification kit (Qiagen). It outlines the basic steps starting from cellular material, lysing and denaturing the sample, and amplifying DNA.
Materials Phi29 DNA Polymerase–Based WGA
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REPLI-g Mini kits (http://www.qiagen.com) containing: Buffer DLB (reconstitute in 500 μl nuclease-free water, mix well, and centrifuge briefly) Current Protocols in Molecular Biology
Stop solution REPLI-g Mini reaction buffer REPLI-g Mini DNA polymerase 1 M DTT (see recipe) Template DNA (cellular material in PBS) Nuclease-free water 0.5-ml microcentrifuge tubes 30°, 65°, and 95°C thermal cycler or water baths 1. Prepare buffer D2 by mixing 11 μl reconstituted buffer DLB and 1 μl of 1 M DTT. This is typically made as a larger cocktail for multiple reactions, and 3.5 μl of buffer D2 is used per sample.
2. Denature the template DNA by placing 3 μl of cellular material (in PBS) into a 0.5-ml microcentrifuge tube. Add 3.5 μl buffer D2 and mix by vortexing. Briefly centrifuge at room temperature and incubate samples 10 min on ice. DNA is alkali denatured during this step. Trace cellular samples in PBS can be amplified with this protocol. Samples cannot be used if they have been treated with formalin or other crosslinking reagents. While amplification from 2000 cells may result in slower amplification kinetics or amplification failure. Blood samples should be mixed with PBS (0.5 μl blood in 2.5 μl PBS). Cellular material can be washed in PBS to eliminate contaminating materials that may inhibit amplification. Every attempt should be made to prevent contamination of the sample and the reaction with DNA from unwanted sources.
3. Neutralize denature buffer by adding 3.5 μl stop solution. Mix by vortexing and centrifuge briefly. 4. Prepare master mix on ice by adding the following reagents in order:
10 μl nuclease-free water 29 μl REPLI-g Mini reaction buffer 1 μl REPLI-g Mini DNA polymerase Mix and centrifuge briefly. Prepare the master mix only in sufficient quantities and immediately prior to use. Keep the master mix on ice and discard any unused portion. The master mix contains all of the components required for DNA amplification and will generate amplification products if exposed to temperatures >4°C for a sufficient time. Even on ice this mixture will be capable of DNA amplification, and it should be combined with sample as quickly as possible. Take precaution to maintain sterility during handling of these stock solutions to prevent contamination with extraneous DNA that could compromise product integrity. Wear gloves at all times during preparation.
5. Assemble amplification reaction by adding 40 μl master mix to 10 μl denatured DNA from blood or cells from step 2. 6. Amplify DNA by incubating 10 to 16 hr at 30°C. During this step the phi29 DNA polymerase extends the random primers, creating new copies of the input genomic DNA while displacing upstream, newly created strands. These displaced strands are in turn primed and amplified. This cascading reaction results in exponential kinetics of amplification proceeding until the available dNTPs are depleted.
7. Inactivate REPLI-g Mini DNA polymerase by heating samples 3 min at 65°C. 8. (Optional) Analyze data (see Support Protocol).
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The product of the reaction can be analyzed by standard agarose gel electrophoresis to confirm that high-molecular-weight DNA has been generated. Typically, the product will migrate as a smear on the gel ranging in size from 3 to 5 kb up to >20 kb. If the precise concentration of product is required, refer to the Support Protocol for methodology to quantify the sample.
9. Store amplified DNA at 4°C for short-term storage (a few weeks), or at –20°C for long-term storage (a few months). For long-term storage beyond 6 months, store at −80°C. SUPPORT PROTOCOL
QUANTIFICATION OF AMPLIFICATION PRODUCTS In some cases, it is helpful to know the amount of amplified material that the wholegenome amplification reaction has produced. Since the product of the reaction is a heterogeneous population of fragments that are a range of sizes generally >2000 bp but 70,000 nt
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Figure 15.13.2 An illustration depicting (A) DNA polymerase initiating replication on a doublestranded segment of DNA. (B) A DNA polymerase with nick translation activity replicates DNA with no net increase in DNA concentration. (C) A DNA polymerase with strand-displacement activity replicates DNA concurrent with a net increase in DNA concentration (amplification). (D) Strand-displacement DNA synthesis is required for rolling circle replication.
Strand displacement DNA synthesis allows a DNA polymerase to synthesize DNA while displacing an upstream strand of DNA. In contrast to nick translation replication of doublestranded DNA by DNA polymerases having associated 5 -3 exonuclease activity (e.g., E. coli DNA Pol I) (Fig. 15.13.2A,B), replication by strand-displacing DNA polymerases results in the net synthesis of DNA (Fig. 15.13.2A,C). This is particularly useful when the polymerase is being used for rolling circle DNA replication (Kornberg and Baker, 1992). Once the enzyme has completed replication of the circular single-stranded DNA
template, strand displacement activity is required to begin the “rolling” mode of amplification (Fig. 15.13.1D). As the size of the input template circle exceeds 75 to 100 nucleotides, strand-displacement DNA synthesis activity becomes an absolute requirement for efficient rolling circle amplification (Kool, 1996). Circles smaller than this can be “rolled” by most any RNA or DNA polymerase due to torsional stress created by requiring the newly made double-stranded DNA to bend sharply. This stress causes the 5 end of the replicated strand to unwind ahead of the replication fork. Phi29 DNA polymerase has no upper limit
The Polymerase Chain Reaction
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Phi29 DNA Polymerase–Based WGA
on the size of DNA being replicated, and in fact performs strand-displacement replication efficiently on linear as well as circular DNA templates. The proofreading activity of phi29 DNA polymerase is specific for degradation of single-stranded DNA and 3 mismatched termini. Inclusion of nuclease resistant phosphorothioate linkages at the 3 ends of primers inhibits their degradation, allowing continued priming events to occur during extended DNA amplification reactions (Dean et al., 2001). This, combined with the observation that due to its high processivity phi29 DNA polymerase can maintain multiple replication forks simultaneously on one DNA template, has led to a remarkable discovery—a simple random-primed DNA synthesis reaction using phi29 DNA polymerase and nuclease-resistant random hexamers can efficiently amplify virtually any DNA template. Unlike PCR, which requires repeated high temperature cycling to generate single-stranded DNA template, amplification reactions with phi29 DNA polymerase use strand-displacement DNA synthesis to continually generate additional single-stranded DNA template as the DNA is replicated in an isothermal reaction. Two additional advantages gained by the use of a DNA polymerase with high processivity for DNA amplification involve replication slippage and strand switching (Kornberg and Baker, 1992). When template sequences are replicated, replication slippage can result in small insertions or deletions, particularly when the template contains short tandem repeats. This is caused by localized DNA melting that occurs while the DNA polymerase is not bound to the 3 end of the growing DNA chain (Fig. 15.13.3). The subsequent annealing of the 3 end of the strand to nearby complementary template sequences allows re-initiation of DNA polymerase replication at an incorrect location (Fig. 15.13.3). Strand switching can also occur when the DNA is not bound by enzyme, but in this case during strand-displacement replication (Fig. 15.13.4A,B). Any inverted repeat of >4 to 5 nucleotides in the template sequence can direct the newly synthesized 3 end to reanneal with the newly displaced strand (Fig. 15.13.4C,D). These events create large inverted repeats in the replication products and lead to terminally amplified products sometimes referred to as “panhandles” in rolling circle type amplification reactions (Lechner et al., 1983). With both replication slippage and strand switching, a paired, extendable 3
end is formed, allowing re-initiation of replication to occur at a new, incorrect template location. Processive DNA polymerases do not dissociate from the replication fork as frequently as distributive DNA polymerases, and consequently, DNA 3 end melting is limited (Canceill et al., 1999). This leads to a reduction in replication errors caused by slippage or strand switching. Phi29 DNA polymerase was used in the development of the method to generically amplify every DNA segment in a sample, termed whole-genome amplification (WGA) or multiple displacement amplification (MDA) (Dean et al., 2001; Lasken et al., 2001). This method takes advantage of the unique properties of Phi29 DNA polymerase and its ability to perform strand displacement DNA synthesis. Using random hexamer priming of DNA, the enzyme catalyzes a cascading reaction whereby input template DNA can be copied repeatedly, with each new strand itself becoming a template for additional amplification. The enzyme has proofreading activity, allowing for high-fidelity products, and reactions can yield >106 -fold amplification, stopping only once the nucleotides are consumed. The method has been used successfully for amplification of trace human DNA samples followed by analysis using virtually every method known (Dean et al., 2002; Lovmar and Syv¨anen, 2006; Spits et al., 2006; Kumar et al., 2008), including for amplification from singlecell bacterial samples (Raghunathan et al., 2005; Marcy et al., 2007), and even singlemolecule samples (Hutchison et al., 2005). There are hundreds of publications describing experiments that have used WGA methods to create DNA products for analysis from trace sources since the method was first published. Non-templated DNA synthesis As an added complication, when extreme care is taken to produce WGA reactions that are free of contaminating DNA, and the reaction is performed with no added input DNA, amplification still proceeds and a nominal amount of product DNA can occasionally be found after incubation. Numerous attempts to clone this product have failed, indicating that in many cases, the product is likely an unusual form of DNA that has been created artificially in these extended reactions. If an extended amplification time is used, DNA yield may not be an indicator of the presence or absence of actual template DNA in the original sample. Trace samples that are allowed to amplify for an extended time can be diluted with
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Figure 15.13.3 An illustration showing how localized DNA melting at the 3 end of a growing DNA strand can result in insertions or deletions due to replication slippage. Thicker lines indicate a short repeated sequence.
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Figure 15.13.4 An Illustration showing how localized DNA melting at the 3 end of a growing DNA strand during strand displacement DNA synthesis can result in strand switching. When an inverted repeat is copied (A), while simultaneously strand-displacing that same sequence (the inverted repeat is shown as a thicker line), a localized 3 end melting can occur if a non-processive polymerase is no longer bound (B). This allows the displaced strand to rebind the template, and for the existing 3 terminus to bind its complement on the displaced strand (C). This can now be extended by polymerase (D), creating a large inverted repeat product due to the polymerase switching from one strand to the other. The Polymerase Chain Reaction
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“junk” DNA made by the DNA polymerase, performing what is called non-templated DNA synthesis. There have been attempts to limit the effect of contamination and junk DNA on product purity (Hutchison et al., 2005; Marcy et al., 2007), but these function by limiting the reaction volumes to decrease the likelihood of contamination and to limit the extent of amplification that can be achieved by 1000fold or more. Non-templated product generation is not a problem if the recommended amount and quality of input template DNA is used.
Phi29 DNA Polymerase–Based WGA
DNA rearrangement during amplification While not a high-frequency occurrence, one issue associated with the WGA method is related to the formation of chimera DNA molecules (Lasken and Stockwell, 2007). In short, it is believed that during these isothermal DNA amplification reactions, an artifactual two-step process occurs. First, the single-stranded intermediate products that are synthesized by strand-displacement synthesis can partially anneal at sites of limited, but significant, short patches of homology. These sections (6- to 12-nt long with 4 to 12 bp of limited homology) are somewhat stable because of the combination of high-ionic-strength buffer conditions that allow for efficient hybridization of the 6-nt random primers used, and the low 30°C temperature used for synthesis by Phi29 DNA polymerase. In the second step, Phi29 DNA polymerase, which is a proofreading enzyme, binds to the free 3 single-stranded end of a newly displaced product strand and degrades the strand with a 3 -5 directionality. This single-stranded DNA degradation activity is found in all proofreading DNA polymerases. When the enzyme reaches one of these short patches of double-stranded homology, it then switches to a DNA synthesis mode, extending the now 3 recessed double-stranded terminus. The resulting product strand is then a chimera product, with the junction sequence having homology to the two different locations that have been artificially joined. There is a suggestion that this process can be stimulated by decreasing the molecular weight of the template DNA used, forcing the reaction to produce more single-stranded displaced 3 termini. Efforts to reduce chimera synthesis have been limited to post-reaction processing (Zhang et al., 2006), and have met with limited success. Some have added bioinformatics processing to manage these chimeric sequences when interpreting DNA sequencing results.
Critical Parameters These methods are sensitive to small amounts of DNA. Wear gloves and safety glasses to avoid contamination. All components should be handled only by persons trained in laboratory techniques, and used in accordance with the principles of good laboratory practice. All chemicals should be considered potentially hazardous; therefore, when handling chemical reagents, it is advisable that suitable protective clothing, such as laboratory coats, safety glasses, and gloves be worn. Care should be taken to avoid contact with skin or eyes. In case of contact with skin or eyes, wash immediately with water. See the appropriate Material Safety Data Sheet for specific recommendations. Use of less DNA or low-quality DNA (such as degraded DNA, or DNA from formalinfixed paraffin embedded samples) can result in poor amplification yield and amplification bias. Unpublished data suggests that underamplification will occur when the target DNA is 100 cells worth of DNA, and ensuring the DNA is of high molecular weight. No amplification product is produced in the absence of template DNA with up to 1.5 hr amplification in typical reactions. However, if amplification reactions are carried out for >1.5 hr, they may produce some artifact DNA synthesis in no-template controls. The enzyme mixtures must be stored at −70°C; all other components may be stored at −20°C. Thaw components on ice and maintain at 0°C to 4°C during handling. If there are doubts about the ability to make DNA-free reagents, all of the reagents needed that are not supplied in the kits described can be purchased from a variety of common vendors, in addition to DNA-free plasticware. Be careful of common laboratory stocks, as these are a frequent source of contamination. When working with samples containing trace DNA, it may be helpful to perform all manipulations in a DNA-free hood.
Troubleshooting Reduced yield/ no amplification product Contamination of template DNA. Excessive contaminants carried over from the starting material can inhibit the DNA polymerase. Dilute or clean-up the DNA and re-amplify. Extending the amplification time will help when inhibitory material is causing reduced yields.
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Inactive enzyme. It is critical that the enzyme be stored properly. Enzyme should be stored at −70°C. If the material will be consumed within 2 months, −20°C storage may be used. The freezer must not be a frost-free unit. Perform a control reaction to confirm performance of the enzyme. Low-quality DNA. Amplification kinetics strongly favors intact templates. Avoid template preparation steps that can damage DNA. Extended vortexing should be avoided. Prolonged denaturation. Heating 3 min at 95°C is sufficient to denature template DNA and facilitate primer annealing. Longer heat denaturing times can nick the template and decrease the amplification efficiency. Poor performance in downstream applications Degraded/low amounts of template DNA. In the absence of input DNA or poor quality of input DNA, there will be no or minimal DNA synthesis in the amplification reactions within 2 hr. Under-representation of subsections of the genome. It has been demonstrated that there is under-amplification near DNA ends. When attempting to perform amplification from 20 kb. As is the case with all DNA amplification methods, there will be an error rate associated with this process; as the extent of DNA amplification is increased, the number of mutations will increase. While this rate is orders of magnitude less than that observed with nonproofreading DNA polymerase-based methods, it can be appreciable and must be considered when interpreting results obtained with DNA produced by these methods. Whole-genome DNA amplification products should be stored and treated as genomic DNA. Be sure to completely mix the sample after thawing, as the DNA can partition when freezing. The described procedures are typically robust when starting with >150 cells worth of genomic DNA (>1 ng). In some cases, the residual buffer may interfere with subsequent manipulations, and in these cases a standard DNA precipitation may be used.
Time Considerations Each of the procedures described in this unit require between 30 and 60 min of handson time, with anywhere from 1.5 to 18 hr of incubation time (depending on the kit and method used).
Literature Cited Blanco, L. and Salas, M. 1984. Characterization and purification of a phage Phi29-encoded DNA polymerase required for the initiation of replication. Proc. Natl. Acad. Sci. U.S.A. 81:53255329. Blanco, L. and Salas, M. 1985. Characterization of a 3 -5 exonuclease activity in the phage Phi29encoded DNA polymerase. Nucleic Acids Res. 13:1239-1249. Blanco, L., Bernad, A., Lazaro, J.M., Martin, G., Garmendia, C., and Salas, M. 1989. Highly efficient DNA synthesis by the phage Phi29 DNA polymerase. Symmetrical mode of DNA replication. J. Biol. Chem. 264:8935-8940. Canceill, D., Viguera, E., and Ehrlich, S.D. 1999. Replication slippage of different DNA polymerases is inversely related to their strand displacement efficiency. J. Biol. Chem. 274:2748127490. de Vega, M., Lazaro, J.M., Salas, M., and Blanco, L. 1996. Primer terminus stabilization at the 3 5 exonuclease active site of Phi29 DNA polymerase. Involvement of two amino acid residues highly conserved in proofreading DNA polymerases. EMBO J. 15:1182-1192. de Vega, M., Ilyina, T., Lazaro, J.M., Salas, M., and Blanco, L. 1997. An invariant lysine residue is involved in catalysis at the 3 -5 exonuclease
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active site of eukaryotic-type DNA polymerases. J. Mol. Biol. 270:65-78. Dean, F.B., Nelson, J.R., Giesler, T.L., and Lasken, R.S. 2001. Rapid amplification of plasmid and phage DNA using Phi29 DNA polymerase and multiply-primed rolling circle amplification. Genome Res. 11:1095-1099. Dean, F.B., Hosono, S., Fang, L., Wu, X., Faruqi, A.F., Bray-Ward, P., Sun, Z., Zong, Q., Du, Y., Du, J., Driscoll, M., Song, W., Kingsmore, S.F., Egholm, M., and Lasken, R.S. 2002. Comprehensive human genome amplification using multiple displacement amplification. Proc. Natl. Acad. Sci. U.S.A. 99:5261-5266. Esteban, J.A., Soengas, M.S., Salas, M., and Blanco, L. 1994. 3 -5 Exonuclease active site of Phi29 DNA polymerase. Evidence favoring a metal ion-assisted reaction mechanism. J. Biol. Chem. 269:31946-31954. Garmendia, C., Bernad, A., Esteban, J.A., Blanco, L., and Salas, M. 1992. The bacteriophage Phi29 DNA polymerase, a proofreading enzyme. J. Biol. Chem. 267:2594-2599. Hutchison, C.A. 3rd, Smith H.O., Pfannkoch, C., and Venter, J.C. 2005. Cell-free cloning using Phi29 DNA polymerase. Proc. Natl. Acad. Sci. U.S.A. 102:17332-17336 Kool, E.T. 1996. Circular oligonucleotides: New concepts in oligonucleotide design. Annu. Rev. Biophys. Biomol. Struct. 25:1-28. Kornberg, A. and Baker, T.A. 1992. DNA Replication. pp. 142-153 and pp. 502-503. W.H. Freeman and Co., New York. Kumar, G., Garnova, E., Reagin, M., and Vidali, A. 2008. Improved multiple displacement amplification with Phi29 DNA polymerase for genotyping of single human cells. Biotechniques 44:879890.
Lechner, R.L., Engler, M.J., and Richardson, C.C. 1983. Characterization of strand displacement synthesis catalyzed by bacteriophage T7 DNA polymerase. J. Biol. Chem. 258:11174-11184. Lovmar, L. and Syv¨anen, A.C. 2006. Multiple displacement amplification to create a long-lasting source of DNA for genetic studies. Hum. Mutat. 27:603-614. Marcy, Y., Ishoey, T., Lasken, R.S., Stockwell, T.B., Walenz, B.P., Halpern, A.L., Beeson, K.Y., Goldberg, S.M., and Quake, S.R. 2007. Nanoliter reactors improve multiple displacement amplification of genomes from single cells. PLoS Genet. 3:1702-1708. Nelson, J.R., Cai, Y.C., Giesler, T.L., Farchaus, J.W., Sundaram, S.T., Ortiz-Rivera, M., Hosta, L.P., Hewitt, P.L., Mamone, J.A., Palaniappan, C., and Fuller, C.W. 2002. TempliPhi, Phi29 DNA polymerase–based rolling circle amplification of templates for DNA sequencing. Biotechniques 32:S44-S47. Raghunathan, A., Ferguson, H.R. Jr., Bornarth, C.J., Song, W., Driscoll, M., and Lasken, R.S. 2005. Genomic DNA amplification from a single bacterium. Appl. Environ. Microbiol. 71:3342-3347. Spits, C., Le Caignec, C., De Rycke, M., Van Haute, L., Van Steirteghem, A., Liebaers, I., and Sermon, K. 2006. Optimization and evaluation of single-cell whole-genome multiple displacement amplification. Hum. Mutat. 27:496-503. Zhang, K., Martiny, A.C., Reppas, N.B., Barry, K.W., Malek, J., Chisholm, S.W., and Church, G.M. 2006. Sequencing genomes from single cells by polymerase cloning. Nat. Biotechnol. 6:680-686.
Internet Resources http://www.gelifesciences.com
Lasken, R.S. and Stockwell, T.B. 2007. Mechanism of chimera formation during the Multiple Displacement Amplification reaction. BMC Biotechnol. 7:19.
For more information on the GenomiPhi product line, new kits and specialty applications, and customized workflows.
Lasken, R.S., Dean, F.B., and Nelson, J.R. 2001. Multiply-primed amplification of nucleic acid sequences. United States Patent 6,323,009.
For more information on the Repli-g product line, new kits and specialty applications, and customized workflows.
http://www.qiagen.com
Phi29 DNA Polymerase–Based WGA
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John R. Nelson1 1
GE Global Research, Niskayuna, New York
ABSTRACT Whole-genome amplification by multiple displacement amplification (MDA) is a patented method to generate potentially unlimited genomic material when researchers are challenged with trace samples, or the amount of genomic DNA required for analysis exceeds the amount on hand. It is an isothermal reaction, using Phi29 DNA polymerase and random hexamer primers for unbiased amplification of linear DNA molecules, such as genomic DNA. The random-primed MDA reaction provides extensive amplification coverage of the genome, generates extremely long DNA products, and provides high DNA yields. This unit explains the reaction, and describes use of the commercial kits C 2014 by John Wiley & Sons, available. Curr. Protoc. Mol. Biol. 105:15.13.1-15.13.16. Inc. Keywords: multiple displacement amplification (MDA) r whole-genome amplification (WGA) r Phi29 DNA polymerase r isothermal amplification
INTRODUCTION The whole-genome DNA amplification method takes advantage of the fact that Phi29 DNA polymerase has excellent strand-displacement activity when replicating DNA (Blanco et al., 1989). Using random hexamer primers to initiate amplification of genomic DNA samples, this single subunit, proofreading DNA polymerase will initiate synthesis at many random loci and, as the template strand is replicated, any doublestranded sections of DNA encountered by the enzyme are displaced off the template. These displaced strands of single-stranded DNA are also randomly bound by primer and replicated in a cascading reaction that typically continues until nucleotides in the reaction mixture are depleted (Fig. 15.13.1). Simple phosphorothioate modification of the random sequence primers prevents the DNA polymerase from degrading the primers and dramatically stimulates reaction kinetics (Dean et al., 2001). The reaction has been shown to provide as much as 106 -fold amplification (Kumar et al., 2008). Large amounts of amplified product DNA can be obtained in just a few hours. Additionally, the presence of an associated proofreading function within the Phi29 DNA polymerase ensures high-fidelity amplification. The error rate of this method has been measured at one in 3 × 105 bases (Nelson et al., 2002). The product DNA can be used directly in most DNA analysis methods as a direct replacement for genomic DNA. In this unit, methods for performing whole-genome amplification using the commercially available kits from both GE (see Basic Protocol 1 and Alternate Protocol 1) and Qiagen (see Basic Protocols 2 and 3) are explained. Basic Protocols 1 and 2 describe utilization of the GenomiPhi DNA Amplification kit and the REPLI-g Mini Kit, respectively, for amplification of purified genomic DNA. Alternate Protocols 1 and 2 describe utilization of the GenomiPhi DNA Amplification kit and the REPLI-g Mini kit, respectively, for amplification of genomic DNA from trace cellular material. The Support Protocol describes The Polymerase Chain Reaction Current Protocols in Molecular Biology 15.13.1-15.13.16, January 2014 Published online January 2014 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/0471142727.mb1513s105 C 2014 John Wiley & Sons, Inc. Copyright
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Figure 15.13.1 Random hexamer primers anneal to the template DNA at multiple sites. Phi29 DNA polymerase initiates replication at multiple sites on the denatured linear DNA simultaneously. As synthesis proceeds, strand displacement of complementary DNA generates new singlestranded DNA. The subsequent priming and strand displacement replication of this DNA results in the formation of double-stranded DNA.
an optional method for the determination of the precise concentration of amplified DNA product in cases where this information is needed. The Commentary section describes the limitations to the protocols and expected results. BASIC PROTOCOL 1
RANDOM-PRIMED, WHOLE-GENOME AMPLIFICATION OF GENOMIC DNA: THE GenomiPhi AMPLIFICATION KIT The purpose of this method is to amplify genomic DNA from any source using the GenomiPhi whole-genome amplification kit (GE Healthcare). It outlines the basic steps starting from purified genomic DNA, denaturing the sample, and amplifying the DNA.
Materials Sample genomic DNA illustra GenomiPhi V2 DNA amplification kit (http://www.gehealthcare.com/lifesciences) containing: Sample buffer Reaction buffer Enzyme mix Control DNA (lambda DNA) TE (see recipe) 0.5-ml plastic microcentrifuge tubes 30°, 65°, and 95°C water baths or thermal cycler 30°C incubator 1. Add 9 μl sample buffer to 1 μl of 1 to 10 ng template DNA. Template DNA should be resuspended in TE or water.
Phi29 DNA Polymerase–Based WGA
Sample genomic DNA should be as intact as possible. Fragmented genomic DNA 3 min or at higher temperatures can cause damage to the DNA.
3. Prepare the master mix. For each amplification reaction, combine 9 μl of reaction buffer with 1 μl enzyme mix on ice. Prepare the master mix only in required quantities and immediately prior to use. Keep the master mix on ice and discard any unused portion. The master mix contains all the components required for DNA amplification and will generate amplification products if exposed to temperatures >4°C for a sufficient time. Even on ice this mixture will be capable of DNA amplification, and it should be combined with sample as quickly as possible. Take precautions to maintain sterility during handling of these stock solutions to prevent contamination with extraneous DNA that could compromise product integrity. Wear gloves at all times during preparation.
4. Assemble amplification reaction by adding 10 μl of prepared master mix from step 3 to each cooled DNA sample, on ice. 5. Amplify DNA by incubating samples 1.5 hr at 30°C. During this step the phi29 DNA polymerase extends the random primers, creating new copies of the input genomic DNA while displacing upstream, newly created strands. These displaced strands are in turn primed and amplified. This cascading reaction results in exponential kinetics of amplification proceeding until the available dNTPs are depleted.
6. Inactivate the Phi29 DNA polymerase enzyme by heating samples 10 min at 65°C and then cooling to 4°C. Heating is required to inactivate the exonuclease activity of the DNA polymerase, which may otherwise begin to degrade the amplification product. The proofreading activity of phi29 DNA polymerase is active on single-stranded DNA, and will degrade single-stranded DNA in a processive manner.
7. (Optional) Analyze the product (see Support Protocol) by standard agarose gel electrophoresis to confirm that high-molecular-weight DNA has been generated. Typically, the product will migrate as a smear on the gel ranging in size from 2 to 5 kb up to >20 kb. If the precise concentration of product is required, see Support Protocol to quantify the sample.
8. Store finished amplification reactions up to 6 months at −20°C. Whole-genome DNA amplification products should be stored and treated as genomic DNA. Be sure to completely mix the sample after thawing, as the DNA can partition when freezing. For long-term storage beyond 6 months, storage at −80°C may be preferred.
WHOLE-GENOME AMPLIFICATION DIRECTLY FROM BLOOD OR CELLS: THE GenomiPhi AMPLIFICATION KIT
ALTERNATE PROTOCOL 1
The purpose of this method is to amplify genomic DNA directly from a small number of cells using the GenomiPhi whole-genome amplification kit (GE Healthcare). It outlines the basic steps starting from cells, lysing the cells while denaturing DNA, and amplifying DNA.
Materials Cell sample in PBS (see recipe for PBS) Denaturation solution (see recipe) Neutralization buffer (see recipe)
The Polymerase Chain Reaction
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GenomiPhi V2 DNA amplification kit (http://www.gehealthcare.com/lifesciences) containing: Sample buffer Reaction buffer Enzyme mix Control DNA (lambda DNA) 0.5-ml microcentrifuge tubes 30°, 65°, and 95°C thermal cycler or water baths 1. Lyse cells and denature genomic DNA by mixing 1 μl cells (suspended in PBS) with 1 μl denaturation solution in a 0.5-ml microcentrifuge tube. Mix by pipetting up and down two to three times. Incubate 10 min on ice. Do not vortex. Trace cellular samples in PBS can be amplified with this protocol. Samples cannot be used if they have been treated with formalin or other crosslinking reagents. While amplification from fewer than 150 cells can be attempted, the resulting amplified DNA may not be high quality. Amplification from >2000 cells may result in slower amplification kinetics or amplification failure. Blood samples should be diluted 1:3 in PBS to dilute inhibitory components. Cellular material can be washed in PBS to eliminate contaminating materials that may inhibit amplification.
2. Neutralize lysed cells/DNA solution by adding 1 μl neutralization buffer and pipetting up and down two to three times. Store on ice. Do not vortex. 3. Prepare the master mix for each amplification reaction. Combine the following for each reaction on ice:
7 μl sample buffer 9 μl reaction buffer 1 μl enzyme mix Prepare the master mix only in required quantities and immediately prior to use. Keep the master mix on ice and discard any unused portion. The master mix contains all of the components required for DNA amplification and will generate amplification products if exposed to temperatures >4°C for sufficient time. Even on ice this mixture will be capable of DNA amplification, and it should be combined with sample as quickly as possible. Take precautions to maintain sterility during handling of these stock solutions to prevent contamination with extraneous DNA that could compromise product integrity. Wear gloves at all times during preparation.
4. Transfer master mix to cooled sample by adding 17 μl of prepared master mix from step 3 to each cooled sample from step 2 on ice. 5. Incubate samples 2 hr at 30°C to amplify DNA. During this step, the phi29 DNA polymerase extends the random primers, creating new copies of the input genomic DNA while displacing upstream, newly created strands. These displaced strands are in turn primed and amplified. This cascading reaction results in exponential kinetics of amplification proceeding until the available dNTPs are depleted.
6. Inactivate Phi29 DNA polymerase enzyme by heating samples 10 min at 65°C and then cooling to 4°C.
Phi29 DNA Polymerase–Based WGA
Heating is required to inactivate the exonuclease activity of the DNA polymerase, which could otherwise begin to degrade the amplification product. The proofreading activity of phi29 DNA polymerase is active on single-stranded DNA, and will processively degrade single-stranded DNA.
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7. (Optional) Analyze product (see Support Protocol). The product of the reaction can be analyzed by standard agarose gel electrophoresis to confirm that high-molecular-weight DNA has been generated. Typically, the product will migrate as a smear on the gel ranging in size from 3 to 5 kb up to >20 kb. If the precise concentration of product is required, refer to the Support Protocol for methodology to quantify the sample.
8. Store completed amplification reactions up to 6 months at −20°C. GenomiPhi DNA V2 amplification products should be stored and treated as genomic DNA. Be sure to completely mix the sample after thawing, as the DNA can partition when freezing. For long-term storage beyond 6 months, storage at −80°C is recommended.
RANDOM-PRIMED, WHOLE-GENOME AMPLIFICATION OF GENOMIC DNA: THE REPLI-g MINI KIT
BASIC PROTOCOL 2
The purpose of this method is to amplify genomic DNA from any source using the REPLI-g mini whole-genome amplification kit (Qiagen). It outlines the basic steps starting from purified genomic DNA, denaturing the sample, and amplifying DNA.
Materials REPLI-g Mini kits (http://www.qiagen.com): Buffer DLB (reconstitute with 500 μl nuclease-free water, mix well, and centrifuge briefly) Stop solution REPLI-g mini reaction buffer REPLI-g mini DNA polymerase Nuclease-free water Template DNA 0.5-ml microcentrifuge tubes Vortex Centrifuge 30°, 65°, and 95°C thermal cycler or water baths 1. Prepare buffer D1 by mixing 0.6 μl of reconstituted buffer DLB and 2 μl nucleasefree water per sample. This is typically made as a larger cocktail for multiple reactions, and 2.5 μl of buffer D1 is used per sample.
2. Prepare buffer N1 by mixing 0.8 μl stop solution and 4.53 μl nuclease-free water per sample. This is typically made as a cocktail for multiple reactions, and 5 μl of buffer N1 is used per sample.
3. Denature template DNA by placing 2.5 μl template DNA into a 0.5-ml microcentrifuge tube. Add 2.5 μl buffer D1 and mix by vortexing. Centrifuge briefly at room temperature and incubate samples 3 min at room temperature (15° to 25°C). DNA is denatured by alkali during this step. This protocol is optimized for whole-genome amplification from >10 ng of purified genomic DNA template. The template DNA should be suspended in TE buffer. Genomic DNA should be as intact as possible. Fragmented genomic DNA 4°C for a sufficient time. Even on ice this mixture will be capable of DNA amplification, and it should be combined with sample as quickly as possible. Take precautions to maintain sterility during handling of these stock solutions to prevent contamination with extraneous DNA that could compromise product integrity. Wear gloves at all times during preparation.
6. Assemble amplification reaction by adding 40 μl master mix to 10 μl denatured DNA from step 5. 7. Incubate 10 to 16 hr at 30°C to amplify DNA. During this step the phi29 DNA polymerase extends the random primers, creating new copies of the input genomic DNA while displacing upstream, newly created strands. These displaced strands are in turn primed and amplified. This cascading reaction results in exponential kinetics of amplification proceeding until the available dNTPs are depleted.
8. Inactivate REPLI-g Mini DNA polymerase by heating samples 3 min at 65°C. Heating is required to inactivate the exonuclease activity of the DNA polymerase, which may otherwise begin to degrade the amplification product. The proofreading activity of phi29 DNA polymerase is active on single-stranded DNA, and will processively degrade single-stranded DNA.
9. (Optional) Analyze the product (see Support Protocol) by standard agarose gel electrophoresis to confirm that high-molecular-weight DNA has been generated. Typically, the product will migrate as a smear on the gel ranging in size from 2 to 5 kb up to >20 kb. If the precise concentration of product is required, see Support Protocol to quantify the sample.
10. Store amplified DNA at 4°C for short-term storage (a few weeks), or –20°C for longer-term storage (a few months). Whole-genome DNA amplification products should be stored and treated as genomic DNA. Be sure to completely mix the sample after thawing, as the DNA can partition when freezing. For long-term storage beyond 6 months, storage at −80°C is recommended. ALTERNATE PROTOCOL 2
RANDOM-PRIMED, WHOLE-GENOME AMPLIFICATION OF GENOMIC DNA FROM BLOOD OR CELLS: THE REPLI-g MINI KIT The purpose of this method is to amplify genomic DNA from trace cells using the REPLIg mini whole-genome amplification kit (Qiagen). It outlines the basic steps starting from cellular material, lysing and denaturing the sample, and amplifying DNA.
Materials Phi29 DNA Polymerase–Based WGA
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REPLI-g Mini kits (http://www.qiagen.com) containing: Buffer DLB (reconstitute in 500 μl nuclease-free water, mix well, and centrifuge briefly) Current Protocols in Molecular Biology
Stop solution REPLI-g Mini reaction buffer REPLI-g Mini DNA polymerase 1 M DTT (see recipe) Template DNA (cellular material in PBS) Nuclease-free water 0.5-ml microcentrifuge tubes 30°, 65°, and 95°C thermal cycler or water baths 1. Prepare buffer D2 by mixing 11 μl reconstituted buffer DLB and 1 μl of 1 M DTT. This is typically made as a larger cocktail for multiple reactions, and 3.5 μl of buffer D2 is used per sample.
2. Denature the template DNA by placing 3 μl of cellular material (in PBS) into a 0.5-ml microcentrifuge tube. Add 3.5 μl buffer D2 and mix by vortexing. Briefly centrifuge at room temperature and incubate samples 10 min on ice. DNA is alkali denatured during this step. Trace cellular samples in PBS can be amplified with this protocol. Samples cannot be used if they have been treated with formalin or other crosslinking reagents. While amplification from 2000 cells may result in slower amplification kinetics or amplification failure. Blood samples should be mixed with PBS (0.5 μl blood in 2.5 μl PBS). Cellular material can be washed in PBS to eliminate contaminating materials that may inhibit amplification. Every attempt should be made to prevent contamination of the sample and the reaction with DNA from unwanted sources.
3. Neutralize denature buffer by adding 3.5 μl stop solution. Mix by vortexing and centrifuge briefly. 4. Prepare master mix on ice by adding the following reagents in order:
10 μl nuclease-free water 29 μl REPLI-g Mini reaction buffer 1 μl REPLI-g Mini DNA polymerase Mix and centrifuge briefly. Prepare the master mix only in sufficient quantities and immediately prior to use. Keep the master mix on ice and discard any unused portion. The master mix contains all of the components required for DNA amplification and will generate amplification products if exposed to temperatures >4°C for a sufficient time. Even on ice this mixture will be capable of DNA amplification, and it should be combined with sample as quickly as possible. Take precaution to maintain sterility during handling of these stock solutions to prevent contamination with extraneous DNA that could compromise product integrity. Wear gloves at all times during preparation.
5. Assemble amplification reaction by adding 40 μl master mix to 10 μl denatured DNA from blood or cells from step 2. 6. Amplify DNA by incubating 10 to 16 hr at 30°C. During this step the phi29 DNA polymerase extends the random primers, creating new copies of the input genomic DNA while displacing upstream, newly created strands. These displaced strands are in turn primed and amplified. This cascading reaction results in exponential kinetics of amplification proceeding until the available dNTPs are depleted.
7. Inactivate REPLI-g Mini DNA polymerase by heating samples 3 min at 65°C. 8. (Optional) Analyze data (see Support Protocol).
The Polymerase Chain Reaction
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The product of the reaction can be analyzed by standard agarose gel electrophoresis to confirm that high-molecular-weight DNA has been generated. Typically, the product will migrate as a smear on the gel ranging in size from 3 to 5 kb up to >20 kb. If the precise concentration of product is required, refer to the Support Protocol for methodology to quantify the sample.
9. Store amplified DNA at 4°C for short-term storage (a few weeks), or at –20°C for long-term storage (a few months). For long-term storage beyond 6 months, store at −80°C. SUPPORT PROTOCOL
QUANTIFICATION OF AMPLIFICATION PRODUCTS In some cases, it is helpful to know the amount of amplified material that the wholegenome amplification reaction has produced. Since the product of the reaction is a heterogeneous population of fragments that are a range of sizes generally >2000 bp but 70,000 nt
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Figure 15.13.2 An illustration depicting (A) DNA polymerase initiating replication on a doublestranded segment of DNA. (B) A DNA polymerase with nick translation activity replicates DNA with no net increase in DNA concentration. (C) A DNA polymerase with strand-displacement activity replicates DNA concurrent with a net increase in DNA concentration (amplification). (D) Strand-displacement DNA synthesis is required for rolling circle replication.
Strand displacement DNA synthesis allows a DNA polymerase to synthesize DNA while displacing an upstream strand of DNA. In contrast to nick translation replication of doublestranded DNA by DNA polymerases having associated 5 -3 exonuclease activity (e.g., E. coli DNA Pol I) (Fig. 15.13.2A,B), replication by strand-displacing DNA polymerases results in the net synthesis of DNA (Fig. 15.13.2A,C). This is particularly useful when the polymerase is being used for rolling circle DNA replication (Kornberg and Baker, 1992). Once the enzyme has completed replication of the circular single-stranded DNA
template, strand displacement activity is required to begin the “rolling” mode of amplification (Fig. 15.13.1D). As the size of the input template circle exceeds 75 to 100 nucleotides, strand-displacement DNA synthesis activity becomes an absolute requirement for efficient rolling circle amplification (Kool, 1996). Circles smaller than this can be “rolled” by most any RNA or DNA polymerase due to torsional stress created by requiring the newly made double-stranded DNA to bend sharply. This stress causes the 5 end of the replicated strand to unwind ahead of the replication fork. Phi29 DNA polymerase has no upper limit
The Polymerase Chain Reaction
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Phi29 DNA Polymerase–Based WGA
on the size of DNA being replicated, and in fact performs strand-displacement replication efficiently on linear as well as circular DNA templates. The proofreading activity of phi29 DNA polymerase is specific for degradation of single-stranded DNA and 3 mismatched termini. Inclusion of nuclease resistant phosphorothioate linkages at the 3 ends of primers inhibits their degradation, allowing continued priming events to occur during extended DNA amplification reactions (Dean et al., 2001). This, combined with the observation that due to its high processivity phi29 DNA polymerase can maintain multiple replication forks simultaneously on one DNA template, has led to a remarkable discovery—a simple random-primed DNA synthesis reaction using phi29 DNA polymerase and nuclease-resistant random hexamers can efficiently amplify virtually any DNA template. Unlike PCR, which requires repeated high temperature cycling to generate single-stranded DNA template, amplification reactions with phi29 DNA polymerase use strand-displacement DNA synthesis to continually generate additional single-stranded DNA template as the DNA is replicated in an isothermal reaction. Two additional advantages gained by the use of a DNA polymerase with high processivity for DNA amplification involve replication slippage and strand switching (Kornberg and Baker, 1992). When template sequences are replicated, replication slippage can result in small insertions or deletions, particularly when the template contains short tandem repeats. This is caused by localized DNA melting that occurs while the DNA polymerase is not bound to the 3 end of the growing DNA chain (Fig. 15.13.3). The subsequent annealing of the 3 end of the strand to nearby complementary template sequences allows re-initiation of DNA polymerase replication at an incorrect location (Fig. 15.13.3). Strand switching can also occur when the DNA is not bound by enzyme, but in this case during strand-displacement replication (Fig. 15.13.4A,B). Any inverted repeat of >4 to 5 nucleotides in the template sequence can direct the newly synthesized 3 end to reanneal with the newly displaced strand (Fig. 15.13.4C,D). These events create large inverted repeats in the replication products and lead to terminally amplified products sometimes referred to as “panhandles” in rolling circle type amplification reactions (Lechner et al., 1983). With both replication slippage and strand switching, a paired, extendable 3
end is formed, allowing re-initiation of replication to occur at a new, incorrect template location. Processive DNA polymerases do not dissociate from the replication fork as frequently as distributive DNA polymerases, and consequently, DNA 3 end melting is limited (Canceill et al., 1999). This leads to a reduction in replication errors caused by slippage or strand switching. Phi29 DNA polymerase was used in the development of the method to generically amplify every DNA segment in a sample, termed whole-genome amplification (WGA) or multiple displacement amplification (MDA) (Dean et al., 2001; Lasken et al., 2001). This method takes advantage of the unique properties of Phi29 DNA polymerase and its ability to perform strand displacement DNA synthesis. Using random hexamer priming of DNA, the enzyme catalyzes a cascading reaction whereby input template DNA can be copied repeatedly, with each new strand itself becoming a template for additional amplification. The enzyme has proofreading activity, allowing for high-fidelity products, and reactions can yield >106 -fold amplification, stopping only once the nucleotides are consumed. The method has been used successfully for amplification of trace human DNA samples followed by analysis using virtually every method known (Dean et al., 2002; Lovmar and Syv¨anen, 2006; Spits et al., 2006; Kumar et al., 2008), including for amplification from singlecell bacterial samples (Raghunathan et al., 2005; Marcy et al., 2007), and even singlemolecule samples (Hutchison et al., 2005). There are hundreds of publications describing experiments that have used WGA methods to create DNA products for analysis from trace sources since the method was first published. Non-templated DNA synthesis As an added complication, when extreme care is taken to produce WGA reactions that are free of contaminating DNA, and the reaction is performed with no added input DNA, amplification still proceeds and a nominal amount of product DNA can occasionally be found after incubation. Numerous attempts to clone this product have failed, indicating that in many cases, the product is likely an unusual form of DNA that has been created artificially in these extended reactions. If an extended amplification time is used, DNA yield may not be an indicator of the presence or absence of actual template DNA in the original sample. Trace samples that are allowed to amplify for an extended time can be diluted with
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Figure 15.13.3 An illustration showing how localized DNA melting at the 3 end of a growing DNA strand can result in insertions or deletions due to replication slippage. Thicker lines indicate a short repeated sequence.
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Figure 15.13.4 An Illustration showing how localized DNA melting at the 3 end of a growing DNA strand during strand displacement DNA synthesis can result in strand switching. When an inverted repeat is copied (A), while simultaneously strand-displacing that same sequence (the inverted repeat is shown as a thicker line), a localized 3 end melting can occur if a non-processive polymerase is no longer bound (B). This allows the displaced strand to rebind the template, and for the existing 3 terminus to bind its complement on the displaced strand (C). This can now be extended by polymerase (D), creating a large inverted repeat product due to the polymerase switching from one strand to the other. The Polymerase Chain Reaction
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“junk” DNA made by the DNA polymerase, performing what is called non-templated DNA synthesis. There have been attempts to limit the effect of contamination and junk DNA on product purity (Hutchison et al., 2005; Marcy et al., 2007), but these function by limiting the reaction volumes to decrease the likelihood of contamination and to limit the extent of amplification that can be achieved by 1000fold or more. Non-templated product generation is not a problem if the recommended amount and quality of input template DNA is used.
Phi29 DNA Polymerase–Based WGA
DNA rearrangement during amplification While not a high-frequency occurrence, one issue associated with the WGA method is related to the formation of chimera DNA molecules (Lasken and Stockwell, 2007). In short, it is believed that during these isothermal DNA amplification reactions, an artifactual two-step process occurs. First, the single-stranded intermediate products that are synthesized by strand-displacement synthesis can partially anneal at sites of limited, but significant, short patches of homology. These sections (6- to 12-nt long with 4 to 12 bp of limited homology) are somewhat stable because of the combination of high-ionic-strength buffer conditions that allow for efficient hybridization of the 6-nt random primers used, and the low 30°C temperature used for synthesis by Phi29 DNA polymerase. In the second step, Phi29 DNA polymerase, which is a proofreading enzyme, binds to the free 3 single-stranded end of a newly displaced product strand and degrades the strand with a 3 -5 directionality. This single-stranded DNA degradation activity is found in all proofreading DNA polymerases. When the enzyme reaches one of these short patches of double-stranded homology, it then switches to a DNA synthesis mode, extending the now 3 recessed double-stranded terminus. The resulting product strand is then a chimera product, with the junction sequence having homology to the two different locations that have been artificially joined. There is a suggestion that this process can be stimulated by decreasing the molecular weight of the template DNA used, forcing the reaction to produce more single-stranded displaced 3 termini. Efforts to reduce chimera synthesis have been limited to post-reaction processing (Zhang et al., 2006), and have met with limited success. Some have added bioinformatics processing to manage these chimeric sequences when interpreting DNA sequencing results.
Critical Parameters These methods are sensitive to small amounts of DNA. Wear gloves and safety glasses to avoid contamination. All components should be handled only by persons trained in laboratory techniques, and used in accordance with the principles of good laboratory practice. All chemicals should be considered potentially hazardous; therefore, when handling chemical reagents, it is advisable that suitable protective clothing, such as laboratory coats, safety glasses, and gloves be worn. Care should be taken to avoid contact with skin or eyes. In case of contact with skin or eyes, wash immediately with water. See the appropriate Material Safety Data Sheet for specific recommendations. Use of less DNA or low-quality DNA (such as degraded DNA, or DNA from formalinfixed paraffin embedded samples) can result in poor amplification yield and amplification bias. Unpublished data suggests that underamplification will occur when the target DNA is 100 cells worth of DNA, and ensuring the DNA is of high molecular weight. No amplification product is produced in the absence of template DNA with up to 1.5 hr amplification in typical reactions. However, if amplification reactions are carried out for >1.5 hr, they may produce some artifact DNA synthesis in no-template controls. The enzyme mixtures must be stored at −70°C; all other components may be stored at −20°C. Thaw components on ice and maintain at 0°C to 4°C during handling. If there are doubts about the ability to make DNA-free reagents, all of the reagents needed that are not supplied in the kits described can be purchased from a variety of common vendors, in addition to DNA-free plasticware. Be careful of common laboratory stocks, as these are a frequent source of contamination. When working with samples containing trace DNA, it may be helpful to perform all manipulations in a DNA-free hood.
Troubleshooting Reduced yield/ no amplification product Contamination of template DNA. Excessive contaminants carried over from the starting material can inhibit the DNA polymerase. Dilute or clean-up the DNA and re-amplify. Extending the amplification time will help when inhibitory material is causing reduced yields.
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Inactive enzyme. It is critical that the enzyme be stored properly. Enzyme should be stored at −70°C. If the material will be consumed within 2 months, −20°C storage may be used. The freezer must not be a frost-free unit. Perform a control reaction to confirm performance of the enzyme. Low-quality DNA. Amplification kinetics strongly favors intact templates. Avoid template preparation steps that can damage DNA. Extended vortexing should be avoided. Prolonged denaturation. Heating 3 min at 95°C is sufficient to denature template DNA and facilitate primer annealing. Longer heat denaturing times can nick the template and decrease the amplification efficiency. Poor performance in downstream applications Degraded/low amounts of template DNA. In the absence of input DNA or poor quality of input DNA, there will be no or minimal DNA synthesis in the amplification reactions within 2 hr. Under-representation of subsections of the genome. It has been demonstrated that there is under-amplification near DNA ends. When attempting to perform amplification from 20 kb. As is the case with all DNA amplification methods, there will be an error rate associated with this process; as the extent of DNA amplification is increased, the number of mutations will increase. While this rate is orders of magnitude less than that observed with nonproofreading DNA polymerase-based methods, it can be appreciable and must be considered when interpreting results obtained with DNA produced by these methods. Whole-genome DNA amplification products should be stored and treated as genomic DNA. Be sure to completely mix the sample after thawing, as the DNA can partition when freezing. The described procedures are typically robust when starting with >150 cells worth of genomic DNA (>1 ng). In some cases, the residual buffer may interfere with subsequent manipulations, and in these cases a standard DNA precipitation may be used.
Time Considerations Each of the procedures described in this unit require between 30 and 60 min of handson time, with anywhere from 1.5 to 18 hr of incubation time (depending on the kit and method used).
Literature Cited Blanco, L. and Salas, M. 1984. Characterization and purification of a phage Phi29-encoded DNA polymerase required for the initiation of replication. Proc. Natl. Acad. Sci. U.S.A. 81:53255329. Blanco, L. and Salas, M. 1985. Characterization of a 3 -5 exonuclease activity in the phage Phi29encoded DNA polymerase. Nucleic Acids Res. 13:1239-1249. Blanco, L., Bernad, A., Lazaro, J.M., Martin, G., Garmendia, C., and Salas, M. 1989. Highly efficient DNA synthesis by the phage Phi29 DNA polymerase. Symmetrical mode of DNA replication. J. Biol. Chem. 264:8935-8940. Canceill, D., Viguera, E., and Ehrlich, S.D. 1999. Replication slippage of different DNA polymerases is inversely related to their strand displacement efficiency. J. Biol. Chem. 274:2748127490. de Vega, M., Lazaro, J.M., Salas, M., and Blanco, L. 1996. Primer terminus stabilization at the 3 5 exonuclease active site of Phi29 DNA polymerase. Involvement of two amino acid residues highly conserved in proofreading DNA polymerases. EMBO J. 15:1182-1192. de Vega, M., Ilyina, T., Lazaro, J.M., Salas, M., and Blanco, L. 1997. An invariant lysine residue is involved in catalysis at the 3 -5 exonuclease
The Polymerase Chain Reaction
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active site of eukaryotic-type DNA polymerases. J. Mol. Biol. 270:65-78. Dean, F.B., Nelson, J.R., Giesler, T.L., and Lasken, R.S. 2001. Rapid amplification of plasmid and phage DNA using Phi29 DNA polymerase and multiply-primed rolling circle amplification. Genome Res. 11:1095-1099. Dean, F.B., Hosono, S., Fang, L., Wu, X., Faruqi, A.F., Bray-Ward, P., Sun, Z., Zong, Q., Du, Y., Du, J., Driscoll, M., Song, W., Kingsmore, S.F., Egholm, M., and Lasken, R.S. 2002. Comprehensive human genome amplification using multiple displacement amplification. Proc. Natl. Acad. Sci. U.S.A. 99:5261-5266. Esteban, J.A., Soengas, M.S., Salas, M., and Blanco, L. 1994. 3 -5 Exonuclease active site of Phi29 DNA polymerase. Evidence favoring a metal ion-assisted reaction mechanism. J. Biol. Chem. 269:31946-31954. Garmendia, C., Bernad, A., Esteban, J.A., Blanco, L., and Salas, M. 1992. The bacteriophage Phi29 DNA polymerase, a proofreading enzyme. J. Biol. Chem. 267:2594-2599. Hutchison, C.A. 3rd, Smith H.O., Pfannkoch, C., and Venter, J.C. 2005. Cell-free cloning using Phi29 DNA polymerase. Proc. Natl. Acad. Sci. U.S.A. 102:17332-17336 Kool, E.T. 1996. Circular oligonucleotides: New concepts in oligonucleotide design. Annu. Rev. Biophys. Biomol. Struct. 25:1-28. Kornberg, A. and Baker, T.A. 1992. DNA Replication. pp. 142-153 and pp. 502-503. W.H. Freeman and Co., New York. Kumar, G., Garnova, E., Reagin, M., and Vidali, A. 2008. Improved multiple displacement amplification with Phi29 DNA polymerase for genotyping of single human cells. Biotechniques 44:879890.
Lechner, R.L., Engler, M.J., and Richardson, C.C. 1983. Characterization of strand displacement synthesis catalyzed by bacteriophage T7 DNA polymerase. J. Biol. Chem. 258:11174-11184. Lovmar, L. and Syv¨anen, A.C. 2006. Multiple displacement amplification to create a long-lasting source of DNA for genetic studies. Hum. Mutat. 27:603-614. Marcy, Y., Ishoey, T., Lasken, R.S., Stockwell, T.B., Walenz, B.P., Halpern, A.L., Beeson, K.Y., Goldberg, S.M., and Quake, S.R. 2007. Nanoliter reactors improve multiple displacement amplification of genomes from single cells. PLoS Genet. 3:1702-1708. Nelson, J.R., Cai, Y.C., Giesler, T.L., Farchaus, J.W., Sundaram, S.T., Ortiz-Rivera, M., Hosta, L.P., Hewitt, P.L., Mamone, J.A., Palaniappan, C., and Fuller, C.W. 2002. TempliPhi, Phi29 DNA polymerase–based rolling circle amplification of templates for DNA sequencing. Biotechniques 32:S44-S47. Raghunathan, A., Ferguson, H.R. Jr., Bornarth, C.J., Song, W., Driscoll, M., and Lasken, R.S. 2005. Genomic DNA amplification from a single bacterium. Appl. Environ. Microbiol. 71:3342-3347. Spits, C., Le Caignec, C., De Rycke, M., Van Haute, L., Van Steirteghem, A., Liebaers, I., and Sermon, K. 2006. Optimization and evaluation of single-cell whole-genome multiple displacement amplification. Hum. Mutat. 27:496-503. Zhang, K., Martiny, A.C., Reppas, N.B., Barry, K.W., Malek, J., Chisholm, S.W., and Church, G.M. 2006. Sequencing genomes from single cells by polymerase cloning. Nat. Biotechnol. 6:680-686.
Internet Resources http://www.gelifesciences.com
Lasken, R.S. and Stockwell, T.B. 2007. Mechanism of chimera formation during the Multiple Displacement Amplification reaction. BMC Biotechnol. 7:19.
For more information on the GenomiPhi product line, new kits and specialty applications, and customized workflows.
Lasken, R.S., Dean, F.B., and Nelson, J.R. 2001. Multiply-primed amplification of nucleic acid sequences. United States Patent 6,323,009.
For more information on the Repli-g product line, new kits and specialty applications, and customized workflows.
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Phi29 DNA Polymerase–Based WGA
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