Additives may also be necessary in the amplification of long target sequences: DMSO often helps in amplifying products of >1kb. Formamide can apparently dramatically improve the specificity of PCR (Sarkar et al., 1990), while glycerol improves the amplification of high (G+C) templates (Smith et al., 1990). 

Polyethylene glycol (PEG) may be a useful additive when DNA template concentration is very low: it promotes macromolecular association by solvent exclusion, meaning the pol can find the DNA.
 

cDNA PCR

A very useful primer for cDNA synthesis and cDNA PCR comes from a sequencing strategy described by Thweatt et al. (1990): this utilised a mixture of three 21-mer primers consisting of 20 T residues with 3'-terminal A, G or C, respectively, to sequence inside the poly(A) region of cDNA clones of mRNA from eukaryotic origin. I have used it to amplify discrete bands from a variety of poly(A)+ virus RNAs, with only a single specific degenerate primer upstream: the T-primer may anneal anywhere in the poly(A) region, but only molecules which anneal at the beginning of the poly(A) tail, and whose 3'-most base is complementary to the base next to the beginning of the tail, will be extended.

eg: 5'-TTTTTTTTTTTTTTTTTTTTTTTTT(A,G,C)-3'

works for amplification of Potyvirus RNA, and eukaryotic mRNA

A simple set of rules for primer sequence design is as follows (adapted from Innis and Gelfand, 1991):

1. primers should be 17-28 bases in length;

2. base composition should be 50-60% (G+C);

3. primers should end (3') in a G or C, or CG or GC: this prevents "breathing" of ends and increases efficiency of priming;

4. Tms between 55-80^oC are preferred;

5. runs of three or more Cs or Gs at the 3'-ends of primers may promote mispriming at G or C-rich sequences (because of stability of annealing), and should be avoided;

6. 3'-ends of primers should not be complementary (ie. base pair), as otherwise primer dimers will be synthesised preferentially to any other product;

7. primer self-complementarity (ability to form 2^o structures such as hairpins) should be avoided.

Examples of inter- and intra-primer complementarity which would result in problems:

Screen shots taken from analyses done using DNAMAN (Lynnon Biosoft, Quebec, Canada).

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