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 Nde I produces condensed bands in a size gradient with good specificity, and therefore is a better choice than a 4 bp cutter such as Hha I. 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 species.