Complete PCR Guide

互联网2013-09-06

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In the polymerase chain reaction (PCR), a thermostable DNA polymerase amplifies DNA that is flanked by known sequences. The known sequences correspond to those on synthetic oligonucleotide primers which are used to initiate the reaction. PCR can be used in many complex ways to achieve different results.

General Issues to Consider:
        Because PCR is very sensitive, product contamination by undesired sequences is problematic. Every effort must made to keep the template free of contaminating, especially previously amplifie d, sequences. Leave the DNA in the refrigerator until all the other reagents are mixed and aliquoted in the reaction tubes. Wear gloves when handling DNA template and PCR reaction mixtures. It is usually easier to make a master mix of everything (including the enzyme - it''s thermal stable) except the template. Aliquot the mixture into tubes, and add the template just prior to putting them in a thermal cycler. Other precautions include the use of cotton plugged pipet tips to reduce aerosol contaminants of amplified products, and separating initial templates from amplified DNA in separate rooms. Negative template controls should be included in all amplifications.
        One of the most critical factors for successful amplification of DNA is the magnesium ion concentration. Too much MgCl₂ will cause high levels of non-specific amplification, while too little will inhibit the reaction. This protocol uses 1.5 mM Mg ++ as the final concentration for Taq DNA polymerase. Use this as a starting point when using previously untested primers and templates. Optimal concentrations may vary from 0.5 to 6 mM and are determined by titration with MgCl 2. Because of this variation, 10X stock solutions frequently have no magnesium in them and the MgCl 2 is added separately. Generally, the dNTP and MgCl 2</ SUB> concentrations are simultaneously adjusted.
        Annealing temperatures are critical and may also require experimental optimization. Low temperature annealing increases non-specific amplification; high temperatures inhibit annealing but may increase specificity. Typical reactions are performed in a range of 55° to 65°C. The temperature optimum for Taq DNA polymerase is 72°C.

Choice of Primers:
        A number of considerations go into designing primers. Primers should not be self-complementary or complementary to each other, especially at their 3'' ends to avoid primer-dimers from forming. Keep the G+C content between 40 and 60%. Avoid long stretches of G+C since they may form secondary structures. Addition of restriction enzyme sites to the 5'' end of the primers serve as useful vehicles for subsequent subcloning.
         Primer concentrations should be in excess of the template throughout the cycling. Typically the primers are used over a 0.1 - 1.0 µM range. Lower concentrations may reduce artifacts and formation of primer-dimers.

Choice of Templates:
        We have successfully used a variety of different templates in amplification reactions. Most human DNA preparations are from fresh peripheral blood leukocytes or cell lines. The cells are lysed, treated with proteanase-K, RNAase-treated, and phenol extracted. The final A260:280 ratio is about 1.8. For amplification of portions of plasmids, we have used everything from CsCl-banded DNA , quick prep DNA , and heat denatured, transformed bacteria. In the latter case, a single bacterial colony (or as small a portion of a frozen stock as possible) was added to 0.5 ml of TE, vortexed, and then centrifuged to pellet the bac teria. The pellet was resuspended or washed once in 100 µl of TE, centrifuged again, and resuspended in 10 µl of TE. The entire sample was added as the DNA template to a total 50 µl reaction volume.

Choice of DNA Polymerases:
        Taq DNA Polymerase - Taq has 5'' to 3'' exonuclease activity, but neither 3'' to 5'' exonuclease nor any endonuclease activity. Its half-life at 95°C is 35-40 minutes, and is 10 minutes at 97.5°C. Molecular weight = 94,000 by SDS-PAGE. GeneAmp 10X PCR buffer II" from Perkin-Elmer Cetus is 500 mM KCl and 100 mM Tris-HCl (pH 8.3). Buffer I had 15 mM MgCl 2 added to it. We dilute the 10X buffer in H 2O; Perkin-Elmer recommends 0.15% NP-40, 0.15% tween-20, 0.1 mM EDTA and 25 mM Tris-HCl, pH 8.3 for AmpliTaq buffer.

        Stoffel fragment - The N-terminal 289 amino acids of Taq were removed to reduce the 5'' to 3'' exonuclease activity. Its half-life at high temperatures is about twice that of Taq DNA polymerase. MgCl 2 concentrations are typically 2-10 mM giving it a broader range of magnesium than Taq. The enzyme is recommended for allele specific PCR, and for amplification of regions with high GC content.

        rTth DNA Polymeras e - This polymerase is used in reverse transcription-PCR where cDNA is first synthesized at 55° - 70°C in the presence of manganese. The buffer contains DMSO and glycerol. One version of this enzyme (rTth DNA polymerase XL) has proofreading activity via its 3'' to 5'' exonuclease activity and is used to generate longer PCR products. Promega states that polymerases with proof reading activities require higher primer concentrations (0.1 - 0.5 µM). The half-life of rTth DNA polymerase is a bout half that of Taq DNA polymerase.

        UlTma DNA Polymerase - has 3'' to 5'' exonuclease, but not 5'' to 3'' exonuclease activity. Its half-life at 97.5°C is 40-50 minutes. 10X Buffer from Perkin-Elmer for this enzyme is 100 mM Tris-HCl, pH 8.8, 100 mM KCl, and 0.02% tween 20. It is used at 2-6 U/100 µl reaction volume. Its fidelity may be higher than other enzymes.

Setting up the Reaction:
For a Single 100 µl PCR Reaction :
    10x PCR buffer                                      10.0 µl
    dNTP mixture (1.25 mM each dNTP)      16.0 µl (final concentration = 200 µM each)
    5'' primer ( 20 µM)                                    5.0 µl (final concentration = 1.0 µM)
    3'' primer (20 µM)                               & nbsp;    5.0 µl (final concentration = 1.0 µM)
    Taq DNA polymerase (5 U/µl stock)          0.5 µl (2.5 units)
    DNA template                                      &nbs p; varies (0.1 to 1.0 µg for genomic DNA;
                                                                 1 ng or less for cloned or amplified DNA)
    Ste rile Water                                          to 100 µl
    Layer 50 - 75 µl of mineral oil on top of reaction mixture.

Sample volumes vary between 25 and 100 µl. Smaller volumes are possible, but larger volumes may reduce yields, perhaps because of the additional time needed to bring the sample to proper temperature. Variation in the concentration of some of the components is acceptable. For example, we have successfully amplified 300 bp regions with 25 µM dNTPs. Primer concentrations may also be reduced to 0.1 µM.

As described above, a "master mix" may be prepared for amplification of DNA in multiple tubes. The following table gives volumes (in µl) for preparation of a master mixture for 1 through 10 reactions of 25 &# 181;l per tube. A slight excess is prepared to provide sufficient volumes for pipeting.

Cycling Parameters:
        To reduce nonspecific amplification, increase the annealing temperature in 3 to 5°C increments. We have used the extension temperature without a separate annealing temperature for particular amplifications with good results.
        "Hot starts" refer to the addition of the polymerase after the DNA has denatured. This should result in minimizi ng nonspecific priming at low temperatures. This is accomplished by heating the sample above the denaturing temperature and then adding the Taq DNA polymerase through the mineral oil layer into the aqueous solution containing the DNA. Wax plugs are also commercially available. They are melted above the sample (lacking polymerase) then allowed to harden at room temperature. Additional reagents can be added above the solid wax (i.e. Taq polymerase), and then the entire sample is added to the thermal cycle r. As the DNA is denatured and the wax melts, the enzyme transfers to the sample to begin amplification.
        A typical cycling temperature profile is:
        94°C for 1 minute
        60°C for 1 minute
        72°C for 1 minute
We use a 5 minutes time period at 94°C to denature template prior to the initial cycle. An old rule of thumb for a 72°C extension temperature is 1 minute for each 1000 base pairs of DNA being amplified. A 2 kb segment would need an extra minute, while segments under 200 bp don''t need an extension plateau. More recently, Perkin Elmer Cetus has reported the extension rates for Taq, Stoffel and rTth polymerases are 2 - 4 kb per minute. Taq DNA polymerase is active over a broad range, not just at the extension temperature. A Perkin-Elmer representative said that they ha ve measured elongation rates of 75 bp/second at 70°C, 24 bp/sec at 55°C, and 1.5 bp/sec at 35°C. Thus, extension occurs throughout the annealing step, further stabilizing the primer-template interactions, but increasing nonspecific products. A final 5 minute incubation at 75#176;C is commonly added at the end of the cycles.

Cloning Amplified Products:
        The ligation efficiency of amplified DNA varies from sequence to sequence. The cause of the problem is either addition of an A at the end of PCR products, and/or Taq polymerase enzyme remaining on the end of the DNA after amplification. Various solutions have been devised to compensate for these problems.
        Perhaps the simplest strategy when you know in advance that you are going to clone the products is to design a restriction enzyme site(s) to the 5'' end of the primers. Be sure these are unique to the primers and are not also present on the sequence you are amplifying. The products are then digested and subcloned into an appropriately digested vector. Note that some enzymes need a certain number of nucleotides beyond their recognition site for digestion.
        In "Improved Cloning Efficiency of Polymerase Chain Reaction (PCR) Products after Proteinase K Digestion" by Crowe et al (1991) Nucl. Acids. Res. 19(1):184, the authors conclude that T aq polymerase sticks to the ends of amplified products. They found that proteinase K treatment increased the yield of their clones.
        Vectors are commercially available that have an overhanging T on them to compensate for the potential overhanging A at the end of some PCR products. We have had many successful ligations of PCR products into blunt-end digested vectors, suggesting that there is a significant proportion of molecules that lack the overhang ing A residue. Others have treated their products for 15 minutes with 10 U of T4 DNA polymerase, or 30-60 minutes with 5-10 units of Klenow to produce blunt end products. For an alternate strategy, see Aslanidis and de Jong, Nucleic Acids Research 18:6069-6074. The method does not use ligation, and reportedly produces only recombinants with high efficiency. Small inserts can give light blue colonies when a pUC-based vector is employed.

Product Fidelity:
    ;     Mutation estimates vary from 10 -3 to10 -5 and vary with the length of the product and the number of cycles. Other parameters have also been found that influence this measurement. To maximize sequence fidelity of the products, or for use in random mutagenesis, the following table may be helpful.

Stock Reagent	1x	2x	3x	4x	5x	6x	7x	8x	9x	10x
25 mM MgCl2 (1.5mM final)	1.5	3.45	4.95	6.45	7.95	10.5	12	13.5	15	16.5
10x Buffer (-Mg)	2.5	5.75	8.25	10.75	13.25	17.5	20	22.5	25	27.5
1 mM ATP & nbsp; (100 mM final)	2.5	5.75	8.25	10.75	13.25	17.5	20	22.5	25	27.5
1 mM GTP (100 mM final)	2.5	5.75	8.25	10.75	13.25	17.5	20	22.5	25	27.5
1 mM TTP (100 mM final)	2.5	5.75	8.25	10.75	13.25	17.5	20	22.5	25	27.5
1 mM CTP (100 mM final)	2.5	5.75	8.25	10.75	13.25	17.5	20	22.5	25	27.5
OR -- for labelling DNA substitute above dCTP with:
1 mM dCTP (cold)	2.1	4.83	6.93	9.03	11.13	14.7	16.8	18.9	21	23.1
32P-dCTP (4µCi)	0.4	0.92	1.32	1.72	2.12	2.8	3.2	3.6	4	4.4
----------------------------
Water	0.7	1.61	2.31	3.01	3.71	4.9	5.6	6.3	7	7.7
20 µM 5'' primer (1 µM final)	1.25	2.875	4.125	5.375	6.625	8.75	10	11.25	12.5	13.75
20 µM 3'' primer (1 µM final)	1.25	2.875	4.125	5.375</ TD>	6.625	8.75	10	11.25	12.5	13.75
Taq Polymerase (5U/µl) (1.5U)	0.3	0.69	0.99	1.29	1.59	2.1	2.4	2.7	3.0	3.3
SUM	17.5	40.25	57.75	75.25	92.7 5	122.5	140	157.5	175	192.5
DNA template: Q.S. with H20 to 7.5 µl	Dispense 17.5 µl of master mix per tube. Add 7.5 µl of DNA to bring final volume to 25 µl. Overlay with 50 µl of mineral oil.

Component	Increased Fidelity	Increased Infidelity
dNTP	Equal concentrations of dNTPs @ 40-50µM each	Unequal concentrations, 1-2 mM of 3 dNTPs with 0.1 - 0.2 mM of the fourth
Mg ++	1 - 1.5 mM	6 - 8 mM with 0.5 mM Mn ++
Temperature	increase	decrease
[Taq]	decrease	increase
Time	decrease extension time	increase extension time < /TD>
# cycles	decrease	increase

Problem Solving Section:

- - (1) No or few products detected
    
    Were all components added?
    
    Was the template denatured?
    
    Are the primers annealing and forming primer dimers?
    
    Are the cycling parameters correct, i.e. low enough for annealing and extension?
    
    (2) Non-specific product amplification
    
    Are the primers specific for the gene of interest?
    
    Was too much polymerase added?
    
    Should the annealing and/or extension temperatures be increased?
    
    Has the Mg⁺⁺ ion concentration been titered? Reducing it may help.
    
    Reduce annealing/extension times, and/or decrease the number of cycles.
    
    Was a non-template DNA negative control run?
    
    (3) Primer-dimer formation
    
    Are the 3'' ends of the primers complementary?
    
    Increase the ratio of template to primers.
    
    Reduce the number of cycles.
    
    Increase the annealing temperature.
    
    (4) Poor restriction enzyme digests of PCR products
    
    The pH of the PCR buffer at 37°C is typically between 8.3 - 8.8, and may be higher than the pH optimum for most restriction enzymes. Promega advises to keep the PCR s olution below 50% of the digest volume.
- - (1) No or few products detected
    
    Were all components added?
    
    Was the template denatured?
    
    Are the primers annealing and forming primer dimers?
    
    Are the cycling parameters correct, i.e. low enough for annealing and extension?
    
    (2) Non-specific product amplification
    
    Are the primers specific for the gene of interest?
    
    Was too much polymerase added?
    
    Should the annealing and/or extension temperatures be increased?
    
    Has the Mg⁺⁺ ion concentration been titered? Reducing it may help.
    
    Reduce annealing/extension times, and/or decrease the number of cycles.
    
    Was a non-template DNA negative control run?
    
    (3) Primer-dimer formation
    
    Are the 3'' ends of the primers complementary?
    
    Increase the ratio of template to primers.
    
    Reduce the number of cycles.
    
    Increase the annealing temperature.
    
    (4) Poor restriction enzyme digests of PCR products
    
    The pH of the PCR buffer at 37°C is typically between 8.3 - 8.8, and may be higher than the pH optimum for most restriction enzymes. Promega advises to keep the PCR s olution below 50% of the digest volume.
- - (1) No or few products detected
    
    Were all components added?
    
    Was the template denatured?
    
    Are the primers annealing and forming primer dimers?
    
    Are the cycling parameters correct, i.e. low enough for annealing and extension?
    
    (2) Non-specific product amplification
    
    Are the primers specific for the gene of interest?
    
    Was too much polymerase added?
    
    Should the annealing and/or extension temperatures be increased?
    
    Has the Mg⁺⁺ ion concentration been titered? Reducing it may help.
    
    Reduce annealing/extension times, and/or decrease the number of cycles.
    
    Was a non-template DNA negative control run?
    
    (3) Primer-dimer formation
    
    Are the 3'' ends of the primers complementary?
    
    Increase the ratio of template to primers.
    
    Reduce the number of cycles.
    
    Increase the annealing temperature.
    
    (4) Poor restriction enzyme digests of PCR products
    
    The pH of the PCR buffer at 37°C is typically between 8.3 - 8.8, and may be higher than the pH optimum for most restriction enzymes. Promega advises to keep the PCR s olution below 50% of the digest volume.
- - (1) No or few products detected
    
    Were all components added?
    
    Was the template denatured?
    
    Are the primers annealing and forming primer dimers?
    
    Are the cycling parameters correct, i.e. low enough for annealing and extension?
    
    (2) Non-specific product amplification
    
    Are the primers specific for the gene of interest?
    
    Was too much polymerase added?
    
    Should the annealing and/or extension temperatures be increased?
    
    Has the Mg⁺⁺ ion concentration been titered? Reducing it may help.
    
    Reduce annealing/extension times, and/or decrease the number of cycles.
    
    Was a non-template DNA negative control run?
    
    (3) Primer-dimer formation
    
    Are the 3'' ends of the primers complementary?
    
    Increase the ratio of template to primers.
    
    Reduce the number of cycles.
    
    Increase the annealing temperature.
    
    (4) Poor restriction enzyme digests of PCR products
    
    The pH of the PCR buffer at 37°C is typically between 8.3 - 8.8, and may be higher than the pH optimum for most restriction enzymes. Promega advises to keep the PCR s olution below 50% of the digest volume.

- - (1) No or few products detected
    
    Were all components added?
    
    Was the template denatured?
    
    Are the primers annealing and forming primer dimers?
    
    Are the cycling parameters correct, i.e. low enough for annealing and extension?
    
    (2) Non-specific product amplification
    
    Are the primers specific for the gene of interest?
    
    Was too much polymerase added?
    
    Should the annealing and/or extension temperatures be increased?
    
    Has the Mg⁺⁺ ion concentration been titered? Reducing it may help.
    
    Reduce annealing/extension times, and/or decrease the number of cycles.
    
    Was a non-template DNA negative control run?
    
    (3) Primer-dimer formation
    
    Are the 3'' ends of the primers complementary?
    
    Increase the ratio of template to primers.
    
    Reduce the number of cycles.
    
    Increase the annealing temperature.
    
    (4) Poor restriction enzyme digests of PCR products
    
    The pH of the PCR buffer at 37°C is typically between 8.3 - 8.8, and may be higher than the pH optimum for most restriction enzymes. Promega advises to keep the PCR s olution below 50% of the digest volume.
- - (1) No or few products detected
    
    Were all components added?
    
    Was the template denatured?
    
    Are the primers annealing and forming primer dimers?
    
    Are the cycling parameters correct, i.e. low enough for annealing and extension?
    
    (2) Non-specific product amplification
    
    Are the primers specific for the gene of interest?
    
    Was too much polymerase added?
    
    Should the annealing and/or extension temperatures be increased?
    
    Has the Mg⁺⁺ ion concentration been titered? Reducing it may help.
    
    Reduce annealing/extension times, and/or decrease the number of cycles.
    
    Was a non-template DNA negative control run?
    
    (3) Primer-dimer formation
    
    Are the 3'' ends of the primers complementary?
    
    Increase the ratio of template to primers.
    
    Reduce the number of cycles.
    
    Increase the annealing temperature.
    
    (4) Poor restriction enzyme digests of PCR products
    
    The pH of the PCR buffer at 37°C is typically between 8.3 - 8.8, and may be higher than the pH optimum for most restriction enzymes. Promega advises to keep the PCR s olution below 50% of the digest volume.
- - (1) No or few products detected
    
    Were all components added?
    
    Was the template denatured?
    
    Are the primers annealing and forming primer dimers?
    
    Are the cycling parameters correct, i.e. low enough for annealing and extension?
    
    (2) Non-specific product amplification
    
    Are the primers specific for the gene of interest?
    
    Was too much polymerase added?
    
    Should the annealing and/or extension temperatures be increased?
    
    Has the Mg⁺⁺ ion concentration been titered? Reducing it may help.
    
    Reduce annealing/extension times, and/or decrease the number of cycles.
    
    Was a non-template DNA negative control run?
    
    (3) Primer-dimer formation
    
    Are the 3'' ends of the primers complementary?
    
    Increase the ratio of template to primers.
    
    Reduce the number of cycles.
    
    Increase the annealing temperature.
    
    (4) Poor restriction enzyme digests of PCR products
    
    The pH of the PCR buffer at 37°C is typically between 8.3 - 8.8, and may be higher than the pH optimum for most restriction enzymes. Promega advises to keep the PCR s olution below 50% of the digest volume.
- - (1) No or few products detected
    
    Were all components added?
    
    Was the template denatured?
    
    Are the primers annealing and forming primer dimers?
    
    Are the cycling parameters correct, i.e. low enough for annealing and extension?
    
    (2) Non-specific product amplification
    
    Are the primers specific for the gene of interest?
    
    Was too much polymerase added?
    
    Should the annealing and/or extension temperatures be increased?
    
    Has the Mg⁺⁺ ion concentration been titered? Reducing it may help.
    
    Reduce annealing/extension times, and/or decrease the number of cycles.
    
    Was a non-template DNA negative control run?
    
    (3) Primer-dimer formation
    
    Are the 3'' ends of the primers complementary?
    
    Increase the ratio of template to primers.
    
    Reduce the number of cycles.
    
    Increase the annealing temperature.
    
    (4) Poor restriction enzyme digests of PCR products
    
    The pH of the PCR buffer at 37°C is typically between 8.3 - 8.8, and may be higher than the pH optimum for most restriction enzymes. Promega advises to keep the PCR s olution below 50% of the digest volume.

Additional Notes on PCR Techniques:

Aliquots of amniotic fluid that have undergone multiple freeze-thaw cycles (which lyses cells) served as an adequate template.

McHale, R.H., P.M. Stapleton and P.L. Bergquist discussed amplification of DNA from tissue samples in "Rapid preparation of blood and tissue samples for polymerase chain reaction", Biotechniques 10: 20-23 (1991).

For RT-PCR reactions, Promega (Promega Notes 62:20, 1997) indicates that about 1 x 10⁵ transcripts are needed to initiate the reaction. If the average mammalian cell has about 10 pg of RNA, 1 µg of total RNA should be fine for amplifying rare transcripts. AMV reverse transcriptase is active at 58°C, while M-MLV RT should not be used above 42°C.

Silver-stained DNA may be amplified as described by Raaphorst, et al, BioT echniques 20:78, 1996.