Chapter 7 The Polymerase Chain Reaction (PCR): General Methods Daniel L.E. Waters and Frances M. Shapter Abstract The polymerase chain reaction (PCR) converts very low quantities of DNA into very high quantities and is the foundation of many specialized techniques of molecular biology. PCR utilizes components of the cellular machinery of mitotic cell division in vitro which respond predictably to user inputs. This chapter introduces the principles of PCR and discusses practical considerations from target sequence definition through to optimization and application. Key words PCR, Template DNA, Taq DNA polymerase, Primer design, Melting temperature
1
Introduction The polymerase chain reaction (PCR) is an integral component of many protocols and is perhaps the key technique of molecular biology. PCR converts very low quantities of DNA into very high quantities which can be used directly or in downstream applications. Following publication of the original method in 1985 [1], the basic PCR procedure has been modified, expanded, and applied to a vast array of problems and techniques, generating a very long list of specialized applications. This chapter introduces and discusses the principles of the generic PCR method. During the process of mitotic cell division, a copy of the genome is created for each new somatic cell. This process doubles the amount of DNA which is shared equally between the two new cells. The doubling of nuclear DNA takes place at each cell division as a multicellular organism is created through continuous cell division starting from the original progenitor cell. The single genome copy in the original single progenitor cell is converted into many billions of copies in the fully mature organism. PCR relies on similar principles and isolated components of this process to convert very low concentrations of DNA into very high concentrations. The primary physical components of PCR are (1) the template DNA, the DNA which is copied; (2) deoxynucleotide triphosphates
Robert J. Henry and Agnelo Furtado (eds.), Cereal Genomics: Methods and Protocols, Methods in Molecular Biology, vol. 1099, DOI 10.1007/978-1-62703-715-0_7, © Springer Science+Business Media New York 2014
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Fig. 1 Schematic diagram of PCR. Starting with a single copy of target DNA sequence, each cycle of PCR (heating/denaturation, cooling/annealing, and extending) doubles the amount of target DNA molecules increasing the concentration of the target DNA exponentially. The number of target DNA molecules increases in the series 1, 2, 4, 8, 16, …, 2n where “n” is the total number of PCR cycles
(dNTPs), the building blocks of DNA. The four nucleotides are adenine triphosphate (ATP), thymine triphosphate (TTP), guanine triphosphate (GTP), and cytosine triphosphate (CTP); (3) Taq DNA polymerase, the enzyme which joins the nucleotides together creating a mirror image of the template; (4) oligonucleotide primers, a sequence of DNA complementary to the target DNA and to which DNA polymerase binds and initiates DNA synthesis; and (5) a buffer solution of appropriate ionic strength and pH. PCR utilizes the heat-stable DNA polymerase Taq DNA polymerase derived from the thermophilic bacterium Thermus aquaticus (Fig. 1). Thermal stability allows the enzyme to withstand the heating required to denature DNA and maintain activity at relatively high temperatures which improves primer specificity. There are three core steps in PCR as follows. Step (1): Denaturation. The PCR tube, a very small test tube, containing the PCR components
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is heated to 94–96 °C. This denatures the DNA, splitting the two complementary strands apart. Step (2): Annealing. The tube is cooled which allows the DNA primers to bind or anneal through base pairing with their target sequence. The primer DNA sequence is complementary to the target. Step (3): Extension. DNA polymerase binds to the DNA primers and then extends the primers one nucleotide at a time, simultaneously reading the template and then placing a nucleotide which complements the template. For example if the template strand has an “A,” DNA polymerase places a “T” in the newly synthesized strand at the complementary position. By repeating this process through a number of cycles, the concentration of the target DNA increases exponentially. If, for example, there was a single copy of target available for amplification, one cycle of PCR will generate a single copy which doubles the amount of target. In the second cycle of PCR there will be two targets which can be copied, doubling the amount of target to four. The third cycle doubles the target from four to eight, then eight to sixteen, and so on. Assuming perfect efficiency of the PCR, there is doubling of the template DNA at each cycle and the final number of target molecules is Y = X(2n) where “X” is the number of targets at cycle one, “n” is the number of cycles, and “Y” is the final number of target molecules. PCR routinely proceeds for 25–35 cycles, and so PCR can multiply the amount of target DNA in the order of 225–235 times. PCR experiments proceed in a number of steps. First, the target sequence needs to be defined and primers designed. Then the PCR is optimized and following optimization applied to the samples of interest.
2
Materials 1. Primer design software. 2. PCR machine. 3. Pipettes and pipette tips (0–20 μL, 0–200 μL, 0–1 mL). 4. 1.5 mL Eppendorf tubes. 5. Thin-walled PCR tubes. 6. Taq polymerase buffer, 10×. 7. Taq polymerase. 8. 50 mM MgCl2. 9. 10 μM Forward primer. 10. Reverse primer. 11. 10 μM dNTPs. 12. Isolated genomic DNA. 13. Sterile deionized water.
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Methods
3.1 PCR Primer Design
After the nature of the DNA sequence within and surrounding the target region, PCR primer quality is the key determinant of how robust any one PCR is. PCR primer design is usually undertaken using PCR primer design software. Many very effective PCR primer design programs are open access and can be found on the Internet. Each piece of software uses an algorithm which gives weight to each of the PCR design parameters. Given the number of parameters which impact upon PCR efficiency and the clear difficulties in accounting for each of them in primer design, primer design software packages are recommended. 1. Primer specificity. There are often several regions of very similar sequence within a genome. Identifying sequences appropriate for primer annealing is nontrivial and a common source of error. Depending on the purpose of the experiment, primers need to anneal to either unique or non-unique regions of the target. If the purpose is to amplify one target sequence, the primers must anneal to regions of the target which are unique to that region if they are not to amplify nontarget, closely related sequences. Intron and intergenic sequences are generally appropriate primer-binding sites for amplification of unique target regions. In contrast, conserved coding sequences shared by a gene family are useful for amplifying members of that gene family. Functional gene regions such as catalytic or substrate-binding domains define gene identity. These regions are highly conserved, and if the target gene is part of a gene family, primers binding to these conserved regions may amplify more than one member of the gene family. 2. Melting temperature (Tm). The temperature at which doublestranded DNA separates and becomes single-stranded DNA is the Tm. Design primer pairs with a Tm of 50–60 °C which is closely matched, ideally within 1 °C of each other (see Note 1). 3. Primer length. The optimal length of PCR primers is around 18–22 bp. Primers of this length achieve the right balance between specificity and appropriate annealing temperature; the longer the primer, the greater the specificity and the higher the annealing temperature. 4. GC content, total, and 3′ end. The total GC content of a primer should be 50 % ± 10 %. Gs and Cs within five nucleotides of the 3' end of primers stabilize primers. More than three Gs or Cs should be avoided within five nucleotides of the 3′ end of primers. A low GC content in the 3′ region minimizes the risk of false priming. 5. Secondary structure. If the 5′ and 3′ ends of a primer are complementary to each other, the 5′ end can loop back and bind to
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the 3′ end of the primer forming a hairpin structure. Similarly, hairpin secondary structure in the target region can seriously interfere with the primer binding. In either case, the yield of PCR product will be reduced. 6. Primer dimers. A primer can bind both to itself or its pair. This needs to be accounted for and avoided, particularly complementary sequences at the 3′ end of primers. 7. Repeat sequence. A repeat sequence within the primer-binding site can lead to mis-priming and should be avoided. Where it is not possible to avoid repeat sequence, they should be kept to a minimum and as a rule of thumb not exceed four. 3.2 Empirical Tests and Optimization
After the primers have been designed and are available for use, the PCR must be empirically tested. 1. Dilute the oligonucleotide primers in sterile deionized water to a working concentration of 10 μM. Mix well and centrifuge. 2. Thaw concentrated stocks of dNTP. After thawing, mix well and centrifuge. In a single Eppendorf tube, mix and dilute in sterile deionized water all four dNTPs so that the final concentration of each dNTP is 2.5 mM. Mix well and centrifuge. 3. Thaw the 10× buffer (−MgCl2) and 50 mM MgCl2 supplied with the Taq polymerase and genomic DNA. After thawing, mix well and centrifuge. 4. Pipette the following components and amounts into two thinwalled PCR tubes (see Notes 2, 3, and 4). Include a notemplate negative control (see Note 5). Mix well and centrifuge (see Note 6).
Component
Test PCR (μL)
Negative control PCR (μL)
10× Buffer (−MgCl2)
2.5
2.5
50 mM MgCl2
1
1
Forward primer 10 μM
1
1
Reverse primer 10 μM
1
1
dNTPs 2.5 mM each
2
2
Taq polymerase
0.2
0.2
Template (genomic DNA) 10 ng/μL
1
0
Water
16.3
17.3
Total
25
25
5. Place the PCR tubes in a thermal cycler, and amplify the target DNA using the following PCR conditions:
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Initial denaturation: −94 °C, 5 min. Denaturation: −94 °C, 30 s. Annealing: −Primer Tm −5 °C, 30 s. Extension: −72 °C, 1 min per 1 kb of PCR fragment length. Final extension: −72 °C for 5 min. 6. Analyze the outcome of the PCR by gel electrophoresis. If the PCR is successful, a band or bands of the expected size of high signal strength will be detected. There should be no band in the negative control lane. 7. In the event of PCR failure, the PCR needs to be optimized. Alteration of any of the PCR parameters, either PCR components or cycling conditions, can affect the final outcome. However, annealing temperature and Mg2+ concentration have the greatest impact. If the annealing temperature is too high or the Mg2+ concentration is too low, the primers do not bind. If the annealing temperature is too low or the Mg2+ concentration too high, the primers bind nonspecifically, amplifying nontarget sequences (see Notes 7–18). 8. Create the following master mix which contains all the components of the PCR with the exception of water and 50 mM MgCl2: Component
(μL)
10× Buffer
55
Forward primer 10 μM
22
Reverse primer 10 μM
22
dNTPs 2.5 mM each
44
Taq polymerase
4.4
Template (genomic DNA) 10 ng/μL
22
Total
169.4
9. Set up five sets of four PCR tubes, each with the following components: Component
Set 1 (μL) Set 2 (μL) Set 3 (μL) Set 4 (μL) Set 5 (μL)
Master mix
7.7
7.7
7.7
7.7
7.7
50 mM MgCl2
0.75
1
1.5
2
2.5
Deionized (MilliQ) 16.55 water
16.3
15.8
15.3
14.8
Total
25
25
25
25
25
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10. Set up a no-template negative control which has the highest concentration of MgCl2:
Component
Negative control PCR (μL)
10× Buffer
2.5
50 mM MgCl2
2.5
Forward primer 10 μM
1
Reverse primer 10 μM
1
dNTPs 2.5 mM each
2
Taq polymerase
0.2
Template (genomic DNA) 10 ng/ μL
0
Deionized (MilliQ) water
14.8
Total
25
11. Program the gradient PCR machine to generate a uniform 50–60 °C temperature gradient across the heating block at the annealing step of the PCR cycle. There is no temperature gradient for the denaturation and annealing steps. Initial denaturation:−94 °C, 5 min. Denaturation: −94 °C, 30 s. Annealing: −50–60 °C gradient, 30 s. Extension: −72 °C, 1 min per 1 kb of length. Final extension: −72 °C for 5 min. 12. Evenly space the four PCR tubes of each set across the heating block of the gradient PCR machine. One tube of each set should be at the lowest temperature (50 °C) and one tube at the highest temperature (60 °C). The negative control tube needs to be positioned at the lowest annealing temperature (50 °C). Run the PCR program. 13. Analyze the PCR products by gel electrophoresis. If the PCR is successful, a band of the expected size and of high signal strength will be detected in at least one lane and no bands in the negative control lane. This will identify which annealing temperature and MgCl2 concentration are appropriate for subsequent experiments. If there are nonspecific amplification products, increase the annealing temperature or decrease the concentration of MgCl2. MgCl2 concentration can be critical. Slight concentration differences can bracket a nonspecific ladder and no PCR product at all. If there is no signal, lower the annealing temperature further or add more MgCl2.
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Notes 1. The annealing temperature can be altered to match the Tm of the PCR primers. However, robust PCR primers accurately amplify target sequence over a wide range of annealing temperatures; many primer pairs designed by PCR primer design software generate the target PCR product using generic PCR solutions and default PCR machine cycling conditions. 2. The extension time of the PCR needs to be increased for longer fragments. Taq polymerase adds nucleotides at a rate of around 2,000/min. An extension time of 30 s is appropriate for PCR products of less than 1 kb in length. 3. The initial denaturation and final extension can be omitted, often without any impact upon the final outcome. Holds of 4 °C post PCR are common but not necessary. DNA is very stable at room temperature, and there is no nuclease activity remaining after PCR cycling. 4. 10 ng/μL of DNA is routinely used for PCR amplification of rice genomic DNA. DNA concentration in a PCR is dependent on genome size; the larger the genome, the greater the quantity of DNA that needs to be added to the PCR. 5. In common with most experiments, a range of positive and negative controls can be included in PCR experiments. These controls are optional with one very important exception: the no template DNA negative control. PCR is very sensitive and can be used to amplify low concentrations of DNA. Once PCR primers have generated PCR product, this product is in relatively very high concentrations and may contaminate subsequent PCR experiments. Particular PCR products may be so problematic that the primer pair for that PCR must be abandoned. In order to detect the presence of contaminating sequences, a no-template DNA negative control must be included in all PCR experiments. Contaminated DNA stocks or test solutions are the most problematic. Ideally, dummy DNA extractions are run through the whole DNA extraction procedure which can be used as PCR negative controls. 6. It is important to ensure that all mixtures are homogeneous. All solutions must be well mixed after thawing, creation of a new solution, and before being mixed with other solutions. 7. The purpose of PCR is to generate high concentrations of DNA sequences of interest. However, high concentrations of DNA pose a risk to subsequent PCR experiments. PCR product can contaminate PCR reagents, including DNA and RNA samples, pipettes, and laboratory surfaces and will give rise to false positives if appropriate steps are not taken. Because of this, it is very important to ensure that PCR products are never
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brought into contact with PCR preparation areas, reagents, or pipettes. If PCR product is to be stored, it should be stored in areas which are separate from genomic DNA or any samples which may be used in PCR. Ideally, material and air flow one way from preparation areas to post-PCR areas. 8. Published papers often include PCR protocols. It is important to remember that PCR protocols are only guidelines. Each laboratory will need to empirically determine the conditions which allow amplification of a particular fragment of DNA in that laboratory. Each PCR machine, DNA polymerase, and DNA sample will behave differently and affect the outcome of the PCR. Practitioners of PCR need to understand the principles of PCR and have the confidence to modify PCR conditions as required. 9. Primers with a high Tm are more likely to undergo stable nonspecific annealing and generate spurious nontarget PCR products. There are three hydrogen bonds between GC pairs and only two between AT. The extra hydrogen bond between the G and C means that GC pairs are more stable than AT pairs. GC content and primer length are the primary determinants of Tm; longer and high GC content primers have higher Tm. 10. Taq polymerase incorporates incorrect bases at a low but significant rate. For many applications including direct sequencing of PCR products this is not important. If, however, DNA sequence is derived from individual molecules arising from PCR, cloned PCR products for example, steps need to be taken to ensure that the sequence is accurate. Addition of proofreading enzymes to the PCR or creating a consensus sequence from several clones minimizes the significance of this issue. The improved accuracy of proofreading enzymes is also useful when PCR products are cloned for protein expression. 11. PCR components are reasonably stable if measures are taken to exclude microbial and nuclease contamination. Primers are an exception. Primers are most stable when stored in high concentration and therefore should not be diluted to working concentrations until needed. Diluting primers to 10× working concentration is a common practice. These should be discarded 1 month after dilution. 12. Native Taq polymerase is active across a wide range of temperatures. If reactions are set up at room temperature, Taq polymerase extends primers which have bound both nonspecifically to the template DNA and to each other. This can lead to the formation of nonspecific PCR products and primer dimers. Traditionally, the response was to minimize Taq polymerase activity by setting up PCRs on ice. The development of chemical- or antibody-inactivated Taq polymerase has circumvented
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this need. There are a number of commercial Taq polymerase preparations in which the enzyme has been inactivated by the addition of a chemical or an antibody. The chemical or the antibody inactivation is removed by the addition of heat. “Hotstart” enzymes such as these generally return higher yields of more specific PCR products and are recommended for most PCR applications. 13. Long-range PCR: PCR is routinely undertaken to amplify DNA fragments ranging from 100 to 5 kbp in length. Longer fragments up to 40 kbp can be amplified, but these are more challenging. Given that long amplicons require long, intact template, DNA quality is particularly important in long-range PCR. Any degradation or physical shearing of the template DNA will reduce amplification efficiency. Long-range PCR often requires enzyme cocktails and additives. The enzyme cocktails include proofreading enzymes such as Pfu. Taq polymerase does not have a proofreading capacity while Pfu DNA polymerase from Pyrococcus furiosus is thermostable and has “proofreading” activity that decreases the error rate by about a fivefold relative to Taq polymerase [2]. Longrange PCR is often employed for tasks where acquisition of DNA sequence is the ultimate goal, and therefore DNA sequence accuracy is important. Mis-incorporated bases can also interfere and stall DNA synthesis which leads to reductions in PCR product yield. 14. Compounds such as DMSO and betaine weaken hydrogen bonding between nucleotides are also utilized in PCR, relaxing secondary structure which may interfere with DNA polymerization. The longer the amplicon, the greater the chance a portion of the target sequence will be difficult to amplify, hence the need for such additives in long-range PCR particularly. Longer primers up to 35 bp are commonly used in long-range PCR; longer primers are more specific and have higher Tm in part to counteract the addition of additives which reduce internucleotide hydrogen bonding. Long templates require long extension times of 15–20 min. 15. PCR enhancer compounds: A number of compounds can be added individually or in combination to improve PCR performance. The compounds include dimethyl sulfoxide (DMSO), N,N,N-trimethylglycine (betaine), formamide, glycerol, nonionic detergents, bovine serum albumin, polyethylene glycol, and tetramethylammonium chloride. Detergents minimize aggregation of polymerase, while the other enhancers generally either minimize template secondary structure which interferes with DNA synthesis and nonspecific primer binding or stabilize Taq polymerase.
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16. Commercial PCR optimization kits which efficiently test many buffer combinations and PCR conditions are available, for a price. If a kit is not used then optimization can proceed in three steps. The first two steps described here apply to most applications. If a PCR primer pair is not successful after completing the first two steps, designing a new primer or primer pair is usually the most efficient means of achieving a positive result. 17. A touch down PCR [3] can be attempted in the event of nonspecific amplification. In a touch down PCR, the annealing temperature reduces at each cycle relative to the previous cycle by a defined amount until the final annealing temperature is reached, which is then maintained until the PCR is completed. 18. The quantities described here are convenient pipetting volumes. PCRs can be run in very small volumes, saving reagent costs. The PCR can be scaled accordingly, maintaining the relative proportions of each component. References 1. Saiki R, Scharf S, Faloona F et al (1985) Enzymatic amplification of beta-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anaemia. Science 230:1350–1354 2. Cline J, Braman JC, Hogrefe HH (1996) PCR fidelity of Pfu DNA polymerase and other ther-
mostable DNA polymerases. Nucleic Acids Res 24:3546–3551 3. Don RH, Cox PT, Wainwright BJ et al (1991) Touchdown PCR to circumvent spurious priming during gene amplification. Nucleic Acids Res 19:4008
1
Introduction The polymerase chain reaction (PCR) is an integral component of many protocols and is perhaps the key technique of molecular biology. PCR converts very low quantities of DNA into very high quantities which can be used directly or in downstream applications. Following publication of the original method in 1985 [1], the basic PCR procedure has been modified, expanded, and applied to a vast array of problems and techniques, generating a very long list of specialized applications. This chapter introduces and discusses the principles of the generic PCR method. During the process of mitotic cell division, a copy of the genome is created for each new somatic cell. This process doubles the amount of DNA which is shared equally between the two new cells. The doubling of nuclear DNA takes place at each cell division as a multicellular organism is created through continuous cell division starting from the original progenitor cell. The single genome copy in the original single progenitor cell is converted into many billions of copies in the fully mature organism. PCR relies on similar principles and isolated components of this process to convert very low concentrations of DNA into very high concentrations. The primary physical components of PCR are (1) the template DNA, the DNA which is copied; (2) deoxynucleotide triphosphates
Robert J. Henry and Agnelo Furtado (eds.), Cereal Genomics: Methods and Protocols, Methods in Molecular Biology, vol. 1099, DOI 10.1007/978-1-62703-715-0_7, © Springer Science+Business Media New York 2014
65
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Daniel L.E. Waters and Frances M. Shapter
Fig. 1 Schematic diagram of PCR. Starting with a single copy of target DNA sequence, each cycle of PCR (heating/denaturation, cooling/annealing, and extending) doubles the amount of target DNA molecules increasing the concentration of the target DNA exponentially. The number of target DNA molecules increases in the series 1, 2, 4, 8, 16, …, 2n where “n” is the total number of PCR cycles
(dNTPs), the building blocks of DNA. The four nucleotides are adenine triphosphate (ATP), thymine triphosphate (TTP), guanine triphosphate (GTP), and cytosine triphosphate (CTP); (3) Taq DNA polymerase, the enzyme which joins the nucleotides together creating a mirror image of the template; (4) oligonucleotide primers, a sequence of DNA complementary to the target DNA and to which DNA polymerase binds and initiates DNA synthesis; and (5) a buffer solution of appropriate ionic strength and pH. PCR utilizes the heat-stable DNA polymerase Taq DNA polymerase derived from the thermophilic bacterium Thermus aquaticus (Fig. 1). Thermal stability allows the enzyme to withstand the heating required to denature DNA and maintain activity at relatively high temperatures which improves primer specificity. There are three core steps in PCR as follows. Step (1): Denaturation. The PCR tube, a very small test tube, containing the PCR components
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is heated to 94–96 °C. This denatures the DNA, splitting the two complementary strands apart. Step (2): Annealing. The tube is cooled which allows the DNA primers to bind or anneal through base pairing with their target sequence. The primer DNA sequence is complementary to the target. Step (3): Extension. DNA polymerase binds to the DNA primers and then extends the primers one nucleotide at a time, simultaneously reading the template and then placing a nucleotide which complements the template. For example if the template strand has an “A,” DNA polymerase places a “T” in the newly synthesized strand at the complementary position. By repeating this process through a number of cycles, the concentration of the target DNA increases exponentially. If, for example, there was a single copy of target available for amplification, one cycle of PCR will generate a single copy which doubles the amount of target. In the second cycle of PCR there will be two targets which can be copied, doubling the amount of target to four. The third cycle doubles the target from four to eight, then eight to sixteen, and so on. Assuming perfect efficiency of the PCR, there is doubling of the template DNA at each cycle and the final number of target molecules is Y = X(2n) where “X” is the number of targets at cycle one, “n” is the number of cycles, and “Y” is the final number of target molecules. PCR routinely proceeds for 25–35 cycles, and so PCR can multiply the amount of target DNA in the order of 225–235 times. PCR experiments proceed in a number of steps. First, the target sequence needs to be defined and primers designed. Then the PCR is optimized and following optimization applied to the samples of interest.
2
Materials 1. Primer design software. 2. PCR machine. 3. Pipettes and pipette tips (0–20 μL, 0–200 μL, 0–1 mL). 4. 1.5 mL Eppendorf tubes. 5. Thin-walled PCR tubes. 6. Taq polymerase buffer, 10×. 7. Taq polymerase. 8. 50 mM MgCl2. 9. 10 μM Forward primer. 10. Reverse primer. 11. 10 μM dNTPs. 12. Isolated genomic DNA. 13. Sterile deionized water.
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Methods
3.1 PCR Primer Design
After the nature of the DNA sequence within and surrounding the target region, PCR primer quality is the key determinant of how robust any one PCR is. PCR primer design is usually undertaken using PCR primer design software. Many very effective PCR primer design programs are open access and can be found on the Internet. Each piece of software uses an algorithm which gives weight to each of the PCR design parameters. Given the number of parameters which impact upon PCR efficiency and the clear difficulties in accounting for each of them in primer design, primer design software packages are recommended. 1. Primer specificity. There are often several regions of very similar sequence within a genome. Identifying sequences appropriate for primer annealing is nontrivial and a common source of error. Depending on the purpose of the experiment, primers need to anneal to either unique or non-unique regions of the target. If the purpose is to amplify one target sequence, the primers must anneal to regions of the target which are unique to that region if they are not to amplify nontarget, closely related sequences. Intron and intergenic sequences are generally appropriate primer-binding sites for amplification of unique target regions. In contrast, conserved coding sequences shared by a gene family are useful for amplifying members of that gene family. Functional gene regions such as catalytic or substrate-binding domains define gene identity. These regions are highly conserved, and if the target gene is part of a gene family, primers binding to these conserved regions may amplify more than one member of the gene family. 2. Melting temperature (Tm). The temperature at which doublestranded DNA separates and becomes single-stranded DNA is the Tm. Design primer pairs with a Tm of 50–60 °C which is closely matched, ideally within 1 °C of each other (see Note 1). 3. Primer length. The optimal length of PCR primers is around 18–22 bp. Primers of this length achieve the right balance between specificity and appropriate annealing temperature; the longer the primer, the greater the specificity and the higher the annealing temperature. 4. GC content, total, and 3′ end. The total GC content of a primer should be 50 % ± 10 %. Gs and Cs within five nucleotides of the 3' end of primers stabilize primers. More than three Gs or Cs should be avoided within five nucleotides of the 3′ end of primers. A low GC content in the 3′ region minimizes the risk of false priming. 5. Secondary structure. If the 5′ and 3′ ends of a primer are complementary to each other, the 5′ end can loop back and bind to
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the 3′ end of the primer forming a hairpin structure. Similarly, hairpin secondary structure in the target region can seriously interfere with the primer binding. In either case, the yield of PCR product will be reduced. 6. Primer dimers. A primer can bind both to itself or its pair. This needs to be accounted for and avoided, particularly complementary sequences at the 3′ end of primers. 7. Repeat sequence. A repeat sequence within the primer-binding site can lead to mis-priming and should be avoided. Where it is not possible to avoid repeat sequence, they should be kept to a minimum and as a rule of thumb not exceed four. 3.2 Empirical Tests and Optimization
After the primers have been designed and are available for use, the PCR must be empirically tested. 1. Dilute the oligonucleotide primers in sterile deionized water to a working concentration of 10 μM. Mix well and centrifuge. 2. Thaw concentrated stocks of dNTP. After thawing, mix well and centrifuge. In a single Eppendorf tube, mix and dilute in sterile deionized water all four dNTPs so that the final concentration of each dNTP is 2.5 mM. Mix well and centrifuge. 3. Thaw the 10× buffer (−MgCl2) and 50 mM MgCl2 supplied with the Taq polymerase and genomic DNA. After thawing, mix well and centrifuge. 4. Pipette the following components and amounts into two thinwalled PCR tubes (see Notes 2, 3, and 4). Include a notemplate negative control (see Note 5). Mix well and centrifuge (see Note 6).
Component
Test PCR (μL)
Negative control PCR (μL)
10× Buffer (−MgCl2)
2.5
2.5
50 mM MgCl2
1
1
Forward primer 10 μM
1
1
Reverse primer 10 μM
1
1
dNTPs 2.5 mM each
2
2
Taq polymerase
0.2
0.2
Template (genomic DNA) 10 ng/μL
1
0
Water
16.3
17.3
Total
25
25
5. Place the PCR tubes in a thermal cycler, and amplify the target DNA using the following PCR conditions:
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Initial denaturation: −94 °C, 5 min. Denaturation: −94 °C, 30 s. Annealing: −Primer Tm −5 °C, 30 s. Extension: −72 °C, 1 min per 1 kb of PCR fragment length. Final extension: −72 °C for 5 min. 6. Analyze the outcome of the PCR by gel electrophoresis. If the PCR is successful, a band or bands of the expected size of high signal strength will be detected. There should be no band in the negative control lane. 7. In the event of PCR failure, the PCR needs to be optimized. Alteration of any of the PCR parameters, either PCR components or cycling conditions, can affect the final outcome. However, annealing temperature and Mg2+ concentration have the greatest impact. If the annealing temperature is too high or the Mg2+ concentration is too low, the primers do not bind. If the annealing temperature is too low or the Mg2+ concentration too high, the primers bind nonspecifically, amplifying nontarget sequences (see Notes 7–18). 8. Create the following master mix which contains all the components of the PCR with the exception of water and 50 mM MgCl2: Component
(μL)
10× Buffer
55
Forward primer 10 μM
22
Reverse primer 10 μM
22
dNTPs 2.5 mM each
44
Taq polymerase
4.4
Template (genomic DNA) 10 ng/μL
22
Total
169.4
9. Set up five sets of four PCR tubes, each with the following components: Component
Set 1 (μL) Set 2 (μL) Set 3 (μL) Set 4 (μL) Set 5 (μL)
Master mix
7.7
7.7
7.7
7.7
7.7
50 mM MgCl2
0.75
1
1.5
2
2.5
Deionized (MilliQ) 16.55 water
16.3
15.8
15.3
14.8
Total
25
25
25
25
25
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10. Set up a no-template negative control which has the highest concentration of MgCl2:
Component
Negative control PCR (μL)
10× Buffer
2.5
50 mM MgCl2
2.5
Forward primer 10 μM
1
Reverse primer 10 μM
1
dNTPs 2.5 mM each
2
Taq polymerase
0.2
Template (genomic DNA) 10 ng/ μL
0
Deionized (MilliQ) water
14.8
Total
25
11. Program the gradient PCR machine to generate a uniform 50–60 °C temperature gradient across the heating block at the annealing step of the PCR cycle. There is no temperature gradient for the denaturation and annealing steps. Initial denaturation:−94 °C, 5 min. Denaturation: −94 °C, 30 s. Annealing: −50–60 °C gradient, 30 s. Extension: −72 °C, 1 min per 1 kb of length. Final extension: −72 °C for 5 min. 12. Evenly space the four PCR tubes of each set across the heating block of the gradient PCR machine. One tube of each set should be at the lowest temperature (50 °C) and one tube at the highest temperature (60 °C). The negative control tube needs to be positioned at the lowest annealing temperature (50 °C). Run the PCR program. 13. Analyze the PCR products by gel electrophoresis. If the PCR is successful, a band of the expected size and of high signal strength will be detected in at least one lane and no bands in the negative control lane. This will identify which annealing temperature and MgCl2 concentration are appropriate for subsequent experiments. If there are nonspecific amplification products, increase the annealing temperature or decrease the concentration of MgCl2. MgCl2 concentration can be critical. Slight concentration differences can bracket a nonspecific ladder and no PCR product at all. If there is no signal, lower the annealing temperature further or add more MgCl2.
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Notes 1. The annealing temperature can be altered to match the Tm of the PCR primers. However, robust PCR primers accurately amplify target sequence over a wide range of annealing temperatures; many primer pairs designed by PCR primer design software generate the target PCR product using generic PCR solutions and default PCR machine cycling conditions. 2. The extension time of the PCR needs to be increased for longer fragments. Taq polymerase adds nucleotides at a rate of around 2,000/min. An extension time of 30 s is appropriate for PCR products of less than 1 kb in length. 3. The initial denaturation and final extension can be omitted, often without any impact upon the final outcome. Holds of 4 °C post PCR are common but not necessary. DNA is very stable at room temperature, and there is no nuclease activity remaining after PCR cycling. 4. 10 ng/μL of DNA is routinely used for PCR amplification of rice genomic DNA. DNA concentration in a PCR is dependent on genome size; the larger the genome, the greater the quantity of DNA that needs to be added to the PCR. 5. In common with most experiments, a range of positive and negative controls can be included in PCR experiments. These controls are optional with one very important exception: the no template DNA negative control. PCR is very sensitive and can be used to amplify low concentrations of DNA. Once PCR primers have generated PCR product, this product is in relatively very high concentrations and may contaminate subsequent PCR experiments. Particular PCR products may be so problematic that the primer pair for that PCR must be abandoned. In order to detect the presence of contaminating sequences, a no-template DNA negative control must be included in all PCR experiments. Contaminated DNA stocks or test solutions are the most problematic. Ideally, dummy DNA extractions are run through the whole DNA extraction procedure which can be used as PCR negative controls. 6. It is important to ensure that all mixtures are homogeneous. All solutions must be well mixed after thawing, creation of a new solution, and before being mixed with other solutions. 7. The purpose of PCR is to generate high concentrations of DNA sequences of interest. However, high concentrations of DNA pose a risk to subsequent PCR experiments. PCR product can contaminate PCR reagents, including DNA and RNA samples, pipettes, and laboratory surfaces and will give rise to false positives if appropriate steps are not taken. Because of this, it is very important to ensure that PCR products are never
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brought into contact with PCR preparation areas, reagents, or pipettes. If PCR product is to be stored, it should be stored in areas which are separate from genomic DNA or any samples which may be used in PCR. Ideally, material and air flow one way from preparation areas to post-PCR areas. 8. Published papers often include PCR protocols. It is important to remember that PCR protocols are only guidelines. Each laboratory will need to empirically determine the conditions which allow amplification of a particular fragment of DNA in that laboratory. Each PCR machine, DNA polymerase, and DNA sample will behave differently and affect the outcome of the PCR. Practitioners of PCR need to understand the principles of PCR and have the confidence to modify PCR conditions as required. 9. Primers with a high Tm are more likely to undergo stable nonspecific annealing and generate spurious nontarget PCR products. There are three hydrogen bonds between GC pairs and only two between AT. The extra hydrogen bond between the G and C means that GC pairs are more stable than AT pairs. GC content and primer length are the primary determinants of Tm; longer and high GC content primers have higher Tm. 10. Taq polymerase incorporates incorrect bases at a low but significant rate. For many applications including direct sequencing of PCR products this is not important. If, however, DNA sequence is derived from individual molecules arising from PCR, cloned PCR products for example, steps need to be taken to ensure that the sequence is accurate. Addition of proofreading enzymes to the PCR or creating a consensus sequence from several clones minimizes the significance of this issue. The improved accuracy of proofreading enzymes is also useful when PCR products are cloned for protein expression. 11. PCR components are reasonably stable if measures are taken to exclude microbial and nuclease contamination. Primers are an exception. Primers are most stable when stored in high concentration and therefore should not be diluted to working concentrations until needed. Diluting primers to 10× working concentration is a common practice. These should be discarded 1 month after dilution. 12. Native Taq polymerase is active across a wide range of temperatures. If reactions are set up at room temperature, Taq polymerase extends primers which have bound both nonspecifically to the template DNA and to each other. This can lead to the formation of nonspecific PCR products and primer dimers. Traditionally, the response was to minimize Taq polymerase activity by setting up PCRs on ice. The development of chemical- or antibody-inactivated Taq polymerase has circumvented
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this need. There are a number of commercial Taq polymerase preparations in which the enzyme has been inactivated by the addition of a chemical or an antibody. The chemical or the antibody inactivation is removed by the addition of heat. “Hotstart” enzymes such as these generally return higher yields of more specific PCR products and are recommended for most PCR applications. 13. Long-range PCR: PCR is routinely undertaken to amplify DNA fragments ranging from 100 to 5 kbp in length. Longer fragments up to 40 kbp can be amplified, but these are more challenging. Given that long amplicons require long, intact template, DNA quality is particularly important in long-range PCR. Any degradation or physical shearing of the template DNA will reduce amplification efficiency. Long-range PCR often requires enzyme cocktails and additives. The enzyme cocktails include proofreading enzymes such as Pfu. Taq polymerase does not have a proofreading capacity while Pfu DNA polymerase from Pyrococcus furiosus is thermostable and has “proofreading” activity that decreases the error rate by about a fivefold relative to Taq polymerase [2]. Longrange PCR is often employed for tasks where acquisition of DNA sequence is the ultimate goal, and therefore DNA sequence accuracy is important. Mis-incorporated bases can also interfere and stall DNA synthesis which leads to reductions in PCR product yield. 14. Compounds such as DMSO and betaine weaken hydrogen bonding between nucleotides are also utilized in PCR, relaxing secondary structure which may interfere with DNA polymerization. The longer the amplicon, the greater the chance a portion of the target sequence will be difficult to amplify, hence the need for such additives in long-range PCR particularly. Longer primers up to 35 bp are commonly used in long-range PCR; longer primers are more specific and have higher Tm in part to counteract the addition of additives which reduce internucleotide hydrogen bonding. Long templates require long extension times of 15–20 min. 15. PCR enhancer compounds: A number of compounds can be added individually or in combination to improve PCR performance. The compounds include dimethyl sulfoxide (DMSO), N,N,N-trimethylglycine (betaine), formamide, glycerol, nonionic detergents, bovine serum albumin, polyethylene glycol, and tetramethylammonium chloride. Detergents minimize aggregation of polymerase, while the other enhancers generally either minimize template secondary structure which interferes with DNA synthesis and nonspecific primer binding or stabilize Taq polymerase.
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16. Commercial PCR optimization kits which efficiently test many buffer combinations and PCR conditions are available, for a price. If a kit is not used then optimization can proceed in three steps. The first two steps described here apply to most applications. If a PCR primer pair is not successful after completing the first two steps, designing a new primer or primer pair is usually the most efficient means of achieving a positive result. 17. A touch down PCR [3] can be attempted in the event of nonspecific amplification. In a touch down PCR, the annealing temperature reduces at each cycle relative to the previous cycle by a defined amount until the final annealing temperature is reached, which is then maintained until the PCR is completed. 18. The quantities described here are convenient pipetting volumes. PCRs can be run in very small volumes, saving reagent costs. The PCR can be scaled accordingly, maintaining the relative proportions of each component. References 1. Saiki R, Scharf S, Faloona F et al (1985) Enzymatic amplification of beta-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anaemia. Science 230:1350–1354 2. Cline J, Braman JC, Hogrefe HH (1996) PCR fidelity of Pfu DNA polymerase and other ther-
mostable DNA polymerases. Nucleic Acids Res 24:3546–3551 3. Don RH, Cox PT, Wainwright BJ et al (1991) Touchdown PCR to circumvent spurious priming during gene amplification. Nucleic Acids Res 19:4008
