CAJ | 학술논문

目的：在灭蚊真菌贵阳腐霉Pr1基因已知序列的基础上，利用锅柄聚合酶链反应法扩增Pr1基因上游未知序列。方法首先采用限制性内切酶BamHⅠ对基因组DNA进行完全酶切，用小牛肠碱性磷酸酶去磷酸化后，与5′磷酸化的寡核苷酸链连接，经过变性、链内退火和聚合酶延伸形成锅柄状，最后进行嵌套式PCR。结果获得了Pr1基因已知序列上游864bp核苷酸序列，GenBank登录号为：JQ975036。结论锅柄聚合酶链反应法可以高度有效地扩增与已知位点相邻的基因组片段，是一种可靠的染色体步移法，有利于Pr1基因全长的克隆。
목적：재멸문진균귀양부매Pr1기인이지서렬적기출상，이용과병취합매련반응법확증Pr1기인상유미지서렬。방법수선채용한제성내절매BamHⅠ대기인조DNA진행완전매절，용소우장감성린산매거린산화후，여5′린산화적과핵감산련련접，경과변성、련내퇴화화취합매연신형성과병상，최후진행감투식PCR。결과획득료Pr1기인이지서렬상유864bp핵감산서렬，GenBank등록호위：JQ975036。결론과병취합매련반응법가이고도유효지확증여이지위점상린적기인조편단，시일충가고적염색체보이법，유리우Pr1기인전장적극륭。
Objective Based on a partial Pr 1 genomic sequence of Pythium guiyangense which has been cloned before ,panhandle polymerase chain reaction ( Panhandle PCR ) strategy was used to amplify the upstream flan-king sequence adjacent to the known sequence of the Pr 1 gene .Methods The genomic DNA was firstly digested with BamHⅠand then treated with calf intestinal alkaline phosphatase ( CIAP).Next,a 5′phosphorylated oligonucleotide was ligated to the 5′ends of BamHⅠ-digested DNA.After denaturation ,intrastrand annealing and polymerase exten-sion,a pan with a handle was formed ,and lastly the nested PCR was performed .Results A 864bp product was am-plified,which was adjacent to the known sequence of Pr 1 gene .The gene has been accessed by GenBank ( Accession:JQ975036 ) .Conclusion Panhandle PCR is a quick and convenient approach for amplifying and identifying un-known partner genes ,which facilitates cloning full-length Pr1 gene .