公开(公告)号：US5665572A

公开(公告)日：1997-09-09

申请号：US294606

申请日：1994-08-23

申请人： Joh-E Ikeda ,  Shinji Hadano ,  Haruhiko Yokoi

发明人： Joh-E Ikeda ,  Shinji Hadano ,  Haruhiko Yokoi

IPC分类号： C12Q1/68 ,  C12P19/34 ,  C07H21/02 ,  C07H21/04

CPC分类号： C12Q1/6832 ,  C12Q1/686

摘要： 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.

摘要翻译： 通过聚合酶链式反应（PCR）扩增模板DNA的方法，其中将具有限制性位点的单个寡核苷酸引物与模板DNA接触，由此寡核苷酸随机退火到模板DNA的单链和与该单链互补的DNA序列 链合成。 初始PCR扩增产生在5'末端具有掺入寡核苷酸序列的合成DNA序列和与模板DNA互补的序列。 在较高严格条件下的第二次PCR扩增扩增模板DNA的区域，得到具有寡核苷酸引物限制性位点的DNA片段。 因此该方法可用于简单高效地扩增未知序列的痕量模板DNA，可用于构建染色体特异性区DNA文库，并开发用于染色体定位的探针。