| 摘要 |
A method of producing a hybrid DNA molecule allowing the assembly of sequenc es x1, x2.....xn where n is greater than or equal to 3 (e.g. give sequences) fr om diverse locations into a hybrid molecule for the purpose of mutation analysi s. The method comprising the steps of: (1) providing in a single reaction mixture: (a) the sequences x1, x2.......xn and their complementary sequences x1', x2'.........xn', to be assembled into the hybrid molecule; (b) for each pair of complementary sequences defined in (a) a respective pair of PCR primers each having a priming sequence and which are such that the primers hybridising to the 3' ends of any two sequences (xi, x'(i+1)), where i is 1 to (n-1), have specifically complementary linker sequences; (2) effecting a fir st stage PCR reaction in which those primers provided with linker sequences are present in limiting concentrations; and (3) effecting a second stage PCR reaction using a single pair of primers one of which provides the 5'-end of the sense strand and other of which provides the 3'-end of the anti-sense strand of the required hybrid molecule; whereby said hybrid molecule is generated.
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