The original goal of the PCR inventors was to develop a new diagnostic technology for the detection of an otherwise hardly traceable virus. The intended amplification procedure should work with tiny amounts of DNA, generating millions of amplicons from a single copy of the virus genome. Optimization of basic PCR protocols for the amplification of specific targets involves several steps:
1) Considerations for the amount of input PCR template: In theory, a single target copy can be amplified (30 PCR cycles will yield up to a billion-fold amplification or femtomole amounts of amplified PCR product). But statistical variations will always result in undesired blank samples, and a threshold of 3-10 copies is a more practical lower limit. But there is also a maximum of input target, which should not exceed 105-106 copies. Apart from rapid consumption of all PCR components, rapid reannealing of both DNA strands leads to kinetic interference with primer hybridization or elongation, and PCR products can act as primers. As a drastic consequence, only a smear of PCR products with divergent sizes will be observed due to premature polymerase termination and staggered priming of PCR products.
2) Primer design: Length should be between 18 and 30 nucleotides with a GC content in the range of 4060%. The following features should be avoided: complementarity of two or more nucleotides at the 3' ends of primer pairs (to reduce formation of primer dimers); T at the 3' end (3'-terminal T increases mismatch tolerance); complementarity within the primers and between primer pairs (detailed help is available from numerous software programs, e.g., the free primer3 at www-genome.wi.mit.edu/genome_ software/other/primer3.html or the commercial Pri-merExpress® from Applied Biosystems).
3) Adjustment of primer annealing temperatures: The simplified formula of Tm [°C]=2x(A:T)+4x(G:C) can be used as a guideline. Whenever possible, use primer pairs with similar Tm values and optimize the PCR protocol by a stepwise increase of the annealing temperature, starting about 5°C below the calculated Tm value.
4) Variety of cycling conditions: In general, three-step amplification cycles are used (Table 1). If high annealing temperatures can be used, two-step amplification cycles are possible (Table 2), and some reports[3,4] suggest that this can result in less premature termination and the formation of more defined PCR products.
5) Optimized concentrations of dNTPs and buffers (especially the divalent cation Mg2+), as well as the use of reaction additives: Some commercial suppliers (e.g., Q-solution from Qiagen with a mixture of monovalent K+ and NH+ ions) offer solutions with a wider window for optimal annealing temperatures, as well as Mg2+ and dNTP concentrations (Table 3).
6) Choice of commercial thermocyclers: A wide range is available, including instruments with several independently operated heating/cooling blocks or with gradient blocks that permit analysis of a temperature series in a single experiment. Blocks are available for your preferred PCR reaction tubes and sample numbers (brief overview in Table 4).
7) ''Hot-start'' PCR or heat-activated DNA polymerase: If highly stringent PCR conditions are desired, consider using ''hot-start'' PCR or heat-activated
I STARTl Heat denaturation: 95°C
Thermostable DNA polymerase
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