PCR仪

Tm = 2(AT) + 4(GC).

The table below shows calculated values for primers of various lengths using this equation, which is known as the Wallace formula, and assuming a 50% GC content4.

Primer Length Tm = 2 (AT) + 4(GC) Primer Length Tm = 2 (AT) + 4(GC)
4 12 °C 22 66 °C
6 18 °C 24 72 °C
8 24 °C 26 78 °C
10 30 °C 28 84 °C
12 36 °C 30 90 °C
14 42 °C 32 96 °C
16 48 °C 34 102 °C
18 54 °C 36 108 °C
20 66 °C 38 114 °C

The temperatures calculated using Wallace's rule are inaccurate at the extremes of this chart.

In addition to calculating the melting temperatures of the primers, care must be taken to ensure that the melting temperature of the product is low enough to ensure 100% melting at 92°C. This parameter will help ensure a more efficient PCR, but is not always necessary for successful PCR. In general, products between 100-600 base pairs are efficiently amplified in many PCR reactions. If there is doubt, the product Tm can be calculated using the formula:

Tm =81.5 + 16.6 (log10[K+] + 0.41 (%G+C)-675/length.

Under standard PCR conditions of 50mM KCL, this reduces to(3):

Tm = 59.9 + 0.41 (%G+C) – 675/length

Specificity
As mentioned above, primer specificity is at least partly dependent on primer length. It is evident that there are many more unique 24 base oligos than there are 15 base pair oligos. That being said, primers must be chosen so that they have a unique sequence within the template DNA that is to be amplified. A primer designed with a highly repetitive sequence will result in a smear when amplifying genomic DNA. However, the same primer may give a single band if a single clone from a genomic library is amplified.

Because Taq Polymerase is active over a broad range of temperatures, primer extension will occur at the lower temperatures of annealing. If the temperature is too low, non-specific priming may occur which can be extended by the polymerase if there is a short homology at the 3' end. In general, a melting temperature of 55 °C - 72 °C gives the best results (Note that this corresponds to a primer length of 18-24 bases using Wallace's rule above).

Complementary Primer Sequences
Primers need to be designed with absolutely no intra-primer homology beyond 3 base pairs. If a primer has such a region of self-homology, “snap back”, partially double-stranded structures, can occur which will interfere with annealing to the template.

Another related danger is inter-primer homology. Partial homology in the middle regions of two primers can interfere with hybridization. If the homology should occur at the 3' end of either primer, Primer dimer formation will occur which, more often than not, will prevent the formation of the desired product via competition.

G/C content and Polypyrimidine (T, C) or polypurine (A, G) stretches
The base composition of primers should be between 45% and 55% GC. The primer sequence must be chosen such that there is no PolyG or Poly C stretches that can promote non-specific annealing. Poly A and Poly T stretches are also to be avoided as these will “breath” and open up stretches of the primer-template complex. This can lower the efficiency of amplification. Polypyrimidine (T, C) and polypurine (A, G) stretches should also be avoided. Ideally the primer will have a near random mix of nucleotides, a 50% GC content and be ~20 bases long. This will put the Tm in the range of 56oC - 62oC1.

3’-end Sequence

It is well established that the 3' terminal position in PCR primers is essential for the control of mis-priming5. We have already explored the problem of primer homologies occurring at these regions. Another variable to look at is the inclusion of a G or C residue at the 3' end of primers. This “GC Clamp” helps to ensure correct binding at the 3' end due to the stronger hydrogen bonding of G/C residues. It also helps to improve the efficiency of the reaction by minimizing any “breathing” that might occur.

Conclusion
It is essential that care is taken in the design of primers for PCR. Several parameters including the length of the primer, %GC content and the 3' sequence need to be optimized for successful PCR. Certain of these parameters can be easily manually optimized while others are best done with commercial computer programs. In any event, careful observance of the general rules of primer design will help ensure successful experiments.

References

Dieffenbach, C.W., Lowe, T.M.J., Dveksler, G.S., General Concepts for PCR Primer Design, in PCR Primer, A Laboratory Manual, Dieffenbach, C.W, and Dveksler, G.S., Ed., Cold Spring Harbor Laboratory Press, New York, 1995, 133-155.
Innis, M.A., and Gelfand, D.H., Optimization of PCRs, in PCR protocols, A Guide to Methods and Applications, Innis, M.A., Gelfand, D.H., Sninsky, J.J., and White, T.J., Ed., CRC Press, London, 1994, 5-11.
Sharrocks, A.D., The design of primers for PCR, in PCR Technology, Current Innovations, Griffin, H.G., and Griffin, A.M, Ed., CRC Press, London, 1994, 5-11.
Suggs, S.V., Hirose, T., Miyake, E.H., Kawashima, M.J., Johnson, K.I., and Wallace, R.B., Using Purified Genes, in ICN-UCLA Symp. Developmental Biology, Vol. 23, Brown, D.D. Ed., Academic Press, New York, 1981, 683.
Kwok, S., Kellog, D.E. McKinney, N., Spasic, D., Goda, L., Levenson, C., and Sninsky, J.J., Effects of primer-template mismatches on the polymerase chain reaction: Human Immunodeficiency Virus 1 model studies. Nucleic Acids Res. 18:999-1005, 1990.
* The Polymerase Chain Reaction (PCR) is protected by patent. The patent is held by Hoffmann-La Roche.

PCR
Polymerase Chain Reaction

1) Add the following to a microfuge tube:
10 ul reaction buffer
1 ul 15 uM forward primer
1 ul 15 uM reverse primer
1 ul template DNA
5 ul 2 mM dNTP
8 ul 25 mM MgCl2 or MgSO4 (volume variable)
water (to make up to 100 ul)
2) Place tube in a thermocycler. Heat sample to 95 °C, then add 0.5 -1 ul of enzyme (Taq, Tli, Pfu etc.). Add a few drops of mineral oil.
3) Start the PCR cycles according the following schemes:
a) denaturation - 94 ° C, 30-90 sec.
b) annealing - 55 °C (or -5° Tm), 0.5-2 min.
c) extension - 72 °C, 1 min. (time depends on length of PCR product and enzyme used)
repeat cycles 29 times
4) Add a final extension step of 5 min. to fill in any uncompleted polymerisation. Then cooled down to 4- 25 °C.

Note:
Most of the parameters can be varied to optimise the PCR (more at Tavi's PCR guide):
a) Mg++ - one of the main variables - change the amount added if the PCR result is poor. Mg++ affects the annealing of the oligo to the template DNA by stabilising the oligo-template interaction, it also stabilises the replication complex of polymerase with template-primer. It can therefore also increases non-specific annealing and produced undesirable PCR products (gives multiple bands in gel). EDTA which chelate Mg++ can change the Mg++ concentration.

b) Template DNA concentration - PCR is very powerful tool for DNA amplification therefore very little DNA is needed. But to reduce the likelihood of error by Taq DNA polymerase, a higher DNA concentration can be used, though too much template may increase the amount of contaminants and reduce efficiency.
c) Enzymes used - Taq DNA polymerase has a higher error rate (no proof-reading 3' to 5' exonuclease activity) than Tli or Pfu. Use Tli, Pfu or other polymerases with good proof-reading capability if high fidelity is needed. Taq, however, is less fussy than other polymerases and less likely to fail. It can be used in combination with other enzymes to increase its fidelity. Taq also tends to add extra A's at the 3'end (extra A's are useful for TA cloning but needs to be removed if blunt end ligation is to be done). More enzymes can also be added to improve efficiency (since Taq may be damaged in repeated cycling) but may increase non-specific PCR products. Vent polymerase may degrade primer and therefore not ideal for mutagenesis-by-PCR work.

d) dNTP - can use up to 1.5 mM dNTP. dNTP chelate Mg++, therefore amount of Mg++ used may need to be changed. However excessive dNTP can increase the error rate and possibly inhibits Taq. Lowering the dNTP (10-50 uM) may therefore also reduce error rate. Larger size PCR fragment need more dNTP.
e) primers - up to 3 uM of primers may be used, but high primer to template ratio can results in non-specific amplification and primer-dimer formation (note: store primers in small aliquots).
f) Primer design - check primer sequences to avoid primer-dimer formation. Add a GC-clamp at the 5' end if a restriction site is introduced there. One or two G or C at the 3' end is fine but try to avoid having too many (it can result in non-specific PCR products). Perfect complementarity of 18 bases or more is ideal. See Guide.
g) Thermal cycling - denaturation time can be increased if template GC content is high. Higher annealing temperature may be needed for primers with high GC content or longer primers (calculate Tm). Using a gradient (if your PCR machine permits it) is a useful way of determining the annealing temperature. Extension time should be extended for larger PCR products; but reduced it whenever possible to limit damage to enzyme. Extension time is also affected by the enzymes used e.g for Taq - assume 1000 base/min (also check suppliers' recommendations, actual rate is much higher). The number of cycle can be increased if the number of template DNA is very low, and decreased if high amount of template DNA is used (higher template DNA is preferable for PCR cloning - lower error rate in the PCR).
h) Additives -
Glycerol (5-10%), formamide (1-5%) or DMSO (2-10%) can be added in PCR for template DNA with high GC content (they change the Tm of primer-template hybridisation reaction and the thermostability of polymerase enzyme). Glycerol can protects Taq against heat damage, while formamide may lower enzyme resistence.
0.5 -2M Betaine (stock solution - 5M) is also useful for PCR over high GC content and long stretches of DNA (Long PCR / LA PCR). Perform a titration to determine to optimum concentration (1.3 M recommended). Reduce melting temperature (92 -93 °C) and annealing temperature (1-2°C lower). It may be useful to use betaine in combination with other reagents like 5%DMSO. Betaine is often the secret (and unnecessarily expensive) ingredient of many commercial kits.
>50mM TMAC (tetramethylammonium chloride), TEAC (tetraethylammonium chloride), and TMANO (trimethlamine N-oxide) can also be used.

BSA (up to 0.8 µg/µl) can also improve efficiency of PCR reaction.
See also Dan Cruickshank's PCR additives and Alkami Enhancers for more.
i) PCR buffer
Higher concentration of PCR buffer may be used to improve efficiency.
This buffer may work better than the buffer supplied from commercial sources.
16.6 mM ammonium sulfate
67.7 mM TRIS-HCl, pH 8.89
10 mM beta-mercaptoethanol
170 micrograms/ml BSA
1.5-3 mM MgCl2