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 91-97oC.
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 95oC, which is partly why one should not do more than about 30 amplification cycles: however, it is possible to reduce the denaturation 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 88oC 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 94oC: 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 96oC for 15 sec.
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
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 5oC 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 1oC 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 55oC, HPV DNA is only specifically amplified at 50oC.
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. (See hybridn.doc).
For example, there is a ? 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:
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 - the answer is that elongation occurs from the moment of annealing, even if this is transient, which results in considerably greater stability. At around 70oC the activity is optimal, and primer extension occurs at up to 100 bases/sec. About 1 min is sufficient for reliable amplification of 2kb sequences (Innis and Gelfand, 1990). Longer products require longer times: 3 min is a good bet for 3kb and longer products. Longer times may also be helpful in later cycles when product concentration exceeds enzyme concentration (>1nM), and when dNTP and / or primer depletion may become limiting.
Recommended buffers generally contain :
10-50mM Tris-HCl pH 8.3,
up to 50mM KCl, 1.5mM or higher MgCl2,
primers 0.2 - 1uM each primer,
50 - 200uM each dNTP,
gelatin or BSA to 100ug/ml,
and/or non-ionic detergents such as Tween-20 or Nonidet P-40 or Triton X-100 (0.05 - 0.10% v/v)
(Innis and Gelfand, 1990). Modern formulations may differ considerably, however - they are also generally proprietary.
PCR is supposed to work well in reverse transcriptase buffer, and vice-versa, meaning 1-tube protocols (with cDNA synthesis and subsequent PCR) are possible (Krawetz et al., 19xx; Fuqua et al., 1990).
Higher than 50mM KCl or NaCl inhibits Taq, but some is necessary to facilitate primer annealing.
[Mg2+] affects primer annealing; Tm of template, product and primer-template associations; product specificity; enzyme activity and fidelity. Taq requires free Mg2+, so allowances should be made for dNTPs, primers and template, all of which chelate and sequester the cation; of these, dNTPs are the most concentrated, so [Mg2+] should be 0.5 - 2.5mM greater than [dNTP]. A titration should be performed with varying [Mg2+] with al