Single Primer ("Semi-Random") PCR

Description

Single primer PCR allows amplification from known to unknown regions in chromosomes, phage, plasmids, large PCR products and other sources of DNA.

At sufficiently low stringency, any primer will misprime while continuing to bind specifically to its intended site. Conditions can usually be found allowing mispriming sufficiently close (<3.5 kb) to the correct site to permit amplification anchored at the same.

Reamplification with a nested primer and the original outside primer generates a product with unique ends. The resulting size shift can be used to diagnose the correct product, which can then be sequenced from either end.

Two methods are presented. The "Short" method can be done quickly and works about 80% of the time in our lab for any given primer Po (see figure below) It has been adapted from Hermann et al (1). The "Long" method, developed in our laboratory (2), requires more time but will often work when the short method fails. Another advantage of the long method is that the product has unique ends, allowing convergent sequencing.

When amplifying out of inverted repeats at the ends of certain elements such as Tn10 or Tn5, gel purify bands before sequencing. If you try to sequence the crude PCR reaction, you will get superimposed sequences coming out of both ends.

In our lab, PCR is always done in an Air ThermoCycler (Idaho Technologies, PO Box 50819, Idaho Falls, ID 83402), which ramps quickly, allowing short dwell times during denaturation and annealing. This enables rapid and stringent polymerization, while minimizing enzyme aging and template hydrolysis. We have not tested the protocal on other machines.

In an appendix at the end, I present some primers which have worked well in our lab when sequencing out of common insertion sequences.