Establishment And Kit Design Of POP-PCR Genome Walking With Different Length Of Overlap

In case of no need for genome-wide sequencing,it is the choice of most laboratories to obtain unknown regions flanking a known sequence by genome walking.At present,many PCR-based genome walking methods have been established.We previously established partially overlapping primer-based PCR(POP-PCR)with 10-bp overlap primers for genome walking.POP-PCR does not require any other operation and is a practical method.Based on our previous studies,we herein established 9-bp and 15-bp overlapping POP-PCR;developed 12-bp overlapping POP-PCR kit and tested its feasibility.The main results are as follows:1.Two sets of POP primers with 9-or 15-bp overlaps were designed.Each set of POP primers consisted of three primers [POP-P(for primary PCR),POP-S(for secondary PCR),POP-T(for tertiary PCR)].The 3'-terminal of POP primer set had a9-or 15-bp consistent sequence(overlap).Because of the short overlap,the3'-terminal overlap of the latter POP primer can only be annealed to the overlap site of the previous PCR product at relatively low temperature.Three primers of each POP primer set were matched with three specific primers for three rounds of nested PCR to obtain a flanking region.The first five high-stringent cycles of each PCR exclusively makes the specific primer bind to its complementary site in known sequence and extend to unknown sequence to form a series of target single strand.In the subsequent low-stringent cycle,the matched POP primer partially anneals to plates,forming a single stranded DNA pool with the POP primer at 5'-terminals,in which 3'-terminal of the target single strand is completely complementary to the specific primers,while the non-target single-strand has no perfect annealing sites for any primers.In the next high-stringent cycle,under the direct of specific primer,the target single strand is transformed into a double strand of which two 3'-terminus complement with specific primer or POP primer,respectively.In the remaining high-stringent cycles,the target DNA molecule is exponentially amplified by the specific primer and POP primer according to the conventional specific amplificationmode;a non-target single strand cannot be transformed into double-stranded DNA due to the lack of complete complementary sites for any primers,and therefore cannot be amplified.The glutamate decarboxylase gene(gadA)locus of Lactobacillus brevis NCL912 and the rice genome inserted with hygromycin gene(hyg)were walked by POP-PCR.The results showed that all POP-PCRs could obtain clear target fragments with an average size of 1.5 kb.2.A 12-bp overlap POP-PCR genome walking kit was designed.The composition and operation of the kit were introduced in detail.The positive control template and human genome template were used to validate the kit.The results showed that the kit could efficiently carry out genome walking and was universal.