An alternative protocol for isolating the flanking sequences of a mutation
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Although spontaneous mutations are of fundamental importance to all aspects of organismal biology, it remains difficult to locate the site of the mutation once a useful mutant has been recovered. Using a site-directed mutation in the promoter of chlorophyll a/b binding protein as a model, we have developed a procedure to tag the mutation site to allow its isolation. In a demonstration of this approach, S1 nuclease mismatch cleavage of the deletion site was used as a model to create a DNA break at the deletion, which was subsequently tagged with a one-nucleotide A-tailing reaction. Ligation-mediated PCR with biotinylated primers allowed the capture of a tagged DNA fragment, leading to the recovery of the flanking sequence of the site of the mutation. The specificity and sensitivity in the real situation were tested by using total genomic DNA of apple variety Wijicik McIntosh as background and titering the mutant site flanking sequence using different amounts of mutant DNA and wild-type DNA. The results showed that the protocol could screen the mutant site in whole genomic DNA in 1 week, but unspecific digestion due to the cross-hybridization between imperfectly matched fragments occurred, and the very low-copy mutation has also yet to be resolved.
KeywordsMutation detection Genomic DNA S1 nuclease Spontaneous mutations
This project was supported by the National Natural Science Foundation (No: 39970526). The authors also wish to thank colleagues in the State Key Laboratories for AgroBiotechnology for their advice and support, especially Master Li-Bing Liu who provided the promoter.