Key techniques and efficiency analysis of amplification of flanking
unknown sequences by inverse PCR
Inverse PCR (IPCR) is an accurate, simple, feasible, and ideal technique
for obtaining unknown sequences. In this study, we used the model
monocot Brachypodium distachyon (ecotype Bd21) to standardize the
conditions and materials required for successfully performing the IPCR.
Analysis of the amplified sequences led us to the following conclusions.
First, the distance between the restriction endonuclease cleavage site
and the unknown–known sequence boundary should be at least 400 bp.
Second, a 6 bp restriction endonuclease such as NdeI produces condensed
bands in a size gradient with good specificity, and therefore is a
better choice than a 4 bp cutter such as HhaI. Third, a distance of
approximately 200 bp between the second primer and the boundary sequence
leads to a better amplification effect and effectively ensures the
integrity of the unknown flanking sequence. The experimental conditions
established in this study serve as a theoretical basis for the
amplification of unknown genome sequences of Gramineae crops and other