DNA Amplification by the Polymerase Chain Reaction: Molecular Biology Made Accessible - Insect Molecular Genetics - page 330

Figure 8.3 Inverse PCR allows amplification of DNA flanking the “core” DNA, for which sequence
information is available. Step 1 involves digesting the template DNA with an appropriate restriction
enzyme to produce fragments ≈ 2-4kb long, with the “core DNA” in the middle. Step 2 involves
circularizing the DNA by ligation. Primers, dNTPs, and DNA polymerase are added and the PCR is
carried out. Primers are oriented so that synthesis of DNA occurs away from the core DNA into the
flanking regions. The PCR product consists of the two flanking regions.
Why not simply use a single DNA polymerase with 3 ′ -exonuclease activ-
ity to edit out the mismatches in extension? Barnes (1994) suggested that the
enzymes with 3 ′ -exonuclease activity could degrade PCR primers, especially
during the long synthesis times. Thus, only small amounts of polymerase with
3 ′ -exonuclease activity should be used. In addition, it is especially important that
the template strands be completely denatured at high temperatures to prevent
renaturation before primers can anneal and be extended ( Cheng 1995 ).
Primer design for Long PCR, as usual, should avoid the potential for second-
ary structure and dimer formation ( Cheng et al. 1994a ). Primers of 21-34 nt that