Tailpcr

This protocol was contributed by John McElver (Syngenta Agribusiness Research Inc., Research Triangle Park).

TAIL-PCR (fhermal asymmetric inter/aced polymerase chain reaction) is a method for amplifying unknown sequences flanking known sequences; for example, the genomic sequences adjacent to the end of a T-DNA element. The technique uses one primer specific to the known sequence and a series of shorter, arbitrary primers that may hybridize to the unknown sequence. Samples are subjected to PCR cycles, alternating between high and low stringency. The first reaction is carried out at high stringency so that the specific primer hybridizes to the sample and DNA synthesis follows. A second, nonspecific cycle follows, allowing hybridization of the shorter primers and synthesis of the complementary strand. Typically, the product of the first reaction is used as a template for a second, and sometimes third, reaction using nested specific primers and the series of nonspecific primers. The protocol below is a modified version of that described by Liu et al. (1995), who first described the application of TAIL-PCR to identification of T-DNA insertion sites.

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