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

Labelling of PCR products with digoxygenin-11-dUTP

(DIG; Roche) need be done only in 50uM each dNTP, with the dTTP substituted to 35% with DIG-11-dUTP. NOTE: that the product will have a higher MW than the native product! This results in a very well labelled probe which can be extensively re-used, for periods up to 3 years. See also here.

Helix Destabilisers / Additives

With NAs of high (G C) content, it may be necessary to use harsher denaturation conditions. For example, one may incorporate up to 10% (w or v/v) :

• dimethyl sulphoxide (DMSO),
• dimethyl formamide (DMF),
• urea
• or formamide

in the reaction mix: these additives are presumed to lower the Tm of the target NA, although DMSO at 10% and higher is known to decrease the activity of Taq by up to 50% (Innis and Gelfand, 1990; Gelfand and White, 1990).

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).