PCR Primer Design And Reaction Optimisation (PCR引物设计及优化反应)

Primer Length

The optimum length of a primer depends upon its (A T) content, and the Tm of its partner if one runs the risk of having problems such as described above. Apart from the Tm, a prime consideration is that the primers should be complex enough so that the likelihood of annealing to sequences other than the chosen target is very low.

For example, there is a ¼ chance (4^-1) of finding an A, G, C or T in any given DNA sequence; there is a 1/16 chance (4^-2) of finding any dinucleotide sequence (eg. AG); a 1/256 chance of finding a given 4-base sequence. Thus, a sixteen base sequence will statistically be present only once in every 4¹⁶ bases (=4 294 967 296, or 4 billion): this is about the size of the human or maize genome, and 1000x greater than the genome size of E. coli. Thus, the association of a greater-than-17-base oligonucleotide with its target sequence is an extremely sequence-specific process, far more so than the specificity of monoclonal antibodies in binding to specific antigenic determinants. Consequently, 17-mer or longer primers are routinely used for amplification from genomic DNA of animals and plants. Too long a primer length may mean that even high annealing temperatures are not enough to prevent mismatch pairing and non-specific priming.

For amplification of cognate sequences from different organisms, or for "evolutionary PCR", one may increase the chances of getting product by designing "degenerate" primers: these would in fact be a set of primers which have a number of options at several positions in the sequence so as to allow annealing to and amplification of a variety of related sequences. For example, Compton (1990) describes using 14-mer primer sets with 4 and 5 degeneracies as forward and reverse primers, respectively, for the amplification of glycoprotein B (gB) from related herpesviruses. The reverse primer sequence was as follows:

TCGAATTCNCCYAAYTGNCCNT

where Y = T C, and N = A G C T, and the 8-base 5'-terminal extension comprises a EcoRI site (underlined) and flanking spacer to ensure the restriction enzyme can cut the product (the New England Biolabs catalogue gives a good list of which enzymes require how long a flanking sequence in order to cut stub ends). Degeneracies obviously reduce the specificity of the primer(s), meaning mismatch opportunities are greater, and background noise increases; also, increased degeneracy means concentration of the individual primers decreases; thus, greater than 512-fold degeneracy should be avoided. However, I have used primers with as high as 256- and 1024-fold degeneracy for the successful amplification and subsequent direct sequencing of a wide range of Mastreviruses against a background of maize genomic DNA (Rybicki and Hughes, 1990).

Primer sequences were derived from multiple sequence alignments; the mismatch positions were used as 4-base degeneracies for the primers (shown as stars; 5 in F and 4 in R), as shown above. Despite their degeneracy, the primers could be used to amplify a 250 bp sequence from viruses differing in sequence by as much as 50% over the target sequence, and 60% overall. They could also be used to very sensitively detect the presence of Maize streak virus DNA against a background of maize genomic DNA, at dilutions as low as 1/10⁹ infected sap / healthy sap (see below).

Some groups use deoxyinosine (dI) at degenerate positions rather than use mixed oligos: this base-pairs with any other base, effectively giving a four-fold degeneracy at any postion in the oligo where it is present. This lessens problems to do with depletion of specific single oligos in a highly degenerate mixture, but may result in too high a degeneracy where there are 4 or more dIs in an oligo.

This is normally 70 - 72oC, for 0.5 - 3 min. Taq actually has a specific activity at 37oC which is very close to that of the Klenow fragment of E coli DNA polymerase I, which accounts for the apparent paradox which results when one tries to understand how primers which anneal at an optimum temperature can then be elongated at a considerably higher temperature