PCR引物设计和优化（PCR PRIMER DESIGN AND REACTION OPTIMISATION ）

Denaturing Temperature and time

The specific complementary association due to hydrogen bonding of single-stranded nucleic acids is referred to as "annealing": two complementary sequences will form hydrogen bonds between their complementary bases ( G to C, and A to T or U ) and form a stable double-stranded, anti-parallel "hybrid" molecule. One may make nucleic acid (NA) single-stranded for the purpose of annealing - if it is not single-stranded already, like most RNA viruses - by heating it to a point above the "melting temperature" of the double- or partially-double-stranded form, and then flash-cooling it: this ensures the "denatured" or separated strands do not re-anneal. Additionally, if the NA is heated in buffers of ionic strength lower than 150mM NaCl , the melting temperature is generally less than 100oC - which is why PCR works with denaturing temperatures of 91-97 ^o C .

A more detailed treatment of annealing / hybridisation is given in an accompanying page, together with explanations of calculations of complexity, conditions for annealing / hybridisation, etc.

Taq polymerase is given as having a half-life of 30 min at 95 ^o C , which is partly why one should not do more than about 30 amplification cycles : however, it is possible to reduce the denatu r ation temperature after about 10 rounds of amplification, as the mean length of target DNA is decreased : for templates of 300bp or less , denaturation temperature may be reduced to as low as 88 ^o C for 50% (G+C) templates (Yap and McGee, 1991), which means one may do as many as 40 cycles without much decrease in enzyme efficiency.

" Time at temperature " is the main reason for denaturation / loss of activity of Taq: thus, if one reduces this, one will increase the number of cycles that are possible , whether the temperature is reduced or not. Normally the denaturation time is 1 min at 94 ^o C : it is possible, for short template sequences, to reduce this to 30 sec or less . Increase in denaturation temperature and decrease in time may also work: Innis and Gelfand (1990) recommend 96 ^o C for 15 sec .

Annealing Temperature and Primer Design

Primer length and sequence are of critical importance in designing the parameters of a successful amplification: the melting temperature of a NA duplex increases both with its length, and with increasing (G+C) content: a simple formula for calculation of the Tm is

Tm = 4(G + C) + 2(A + T) ^o C.

Thus, the annealing temperature chosen for a PCR depends directly on length and composition of the primer(s). One should aim at using an annealing temperature (Ta) about 5 ^o C below the lowest Tm of ther pair of primers to be used (Innis and Gelfand, 1990). A more rigorous treatment of Ta is given by Rychlik et al . (1990): they maintain that if the Ta is increased by 1 ^o C every other cycle, specificity of amplification and yield of products <1kb in length are both increased. One consequence of having too low a Ta is that one or both primers will anneal to sequences other than the true target, as internal single-base mismatches or partial annealing may be tolerated: this is fine if one wishes to amplify similar or related targets ; however, it can lead to "non-specific" amplification and consequent reduction in yield of the desired product, if the 3"-most base is paired with a target.

A consequence of too high a Ta is that too little product will be made , as the likelihood of primer annealing is reduced; another and important consideration is that a pair of primers with very different Tas may never give appreciable yields of a unique product, and may also result in inadvertent " asymmetric " or single-strand amplification of the most efficiently primed product strand.

Annealing does not take long: most primers will anneal efficiently in 30 sec or less , unless the Ta is too close to the Tm, or unless they are unusually long.

An illustration of the effect of annealing temperature on the specificity and on the yield of amplification of Human papillomavirus type 16 (HPV-16) is given below (Williamson and Rybicki, 1991: J Med Virol 33: 165-171).

Plasmid and biopsy sample DNA templates were amplified at different annealing temperatures as shown: note that while plasmid is amplified from 37 to 55 ^o C, HPV DNA is only specifically amplified at 50 ^o C.