Research Development Corporation of Japan

Haruhiko Yokoi

Joh-E Ikeda

Shinji Hadano

A method of amplifying template DNA by polymerase chain reaction (PCR) in which a single oligonucleotide primer having a restriction site is contacted with the template DNA, whereby the oligonucleotide randomly anneals to a single strand of the template DNA and DNA sequences complementary to the single strand are synthesized. An initial PCR amplification yields synthetic DNA sequences having the oligonucleotide sequence incorporated therein at the 5' end, and a sequence complementary to the template DNA. A second PCR amplification under higher stringency conditions amplifies regions of the template DNA to give DNA fragments having the restriction sites of the oligonucleotide primer. Thereby the method can be used to amplify trace quantities of template DNA of unknown sequence simply and efficiently, which has applications in the construction of DNA libraries of chromosome specific regions and the development of probes for chromosome mapping.

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Kanagawa, Japan

Haruhiko Yokoi