5' AND 3' POLYMERASE CHAIN REACTION WALKING FROM KNOWN DNA SEQUENCES

摘要

The present invention is directed to a method of polymerase chain reaction (PCR) walking for generating vector free libraries of genomic DNA from which flanking sequences of known DNA sequences can be cloned and amplified. This method of PCR walking involves using unphosphorylated or phosphorylated DNA linkers which will ligate only at the 5' ends, blocking the unligated 3' ends, and specifically priming the synthesis of the desired flanking sequence with a primer complementary to the known DNA sequence. Cloning DNA sequences located 5' to the zeta-globin promoter region by using a polymerase chain reaction walking library is presented as an example.