PCR筛选BAC文库和直接BAC末端测序方法的建立_英文_.pdfVIP

　遗 传 学 报 　Act a Genetica Sinica , November 2004 , 3 1 ( 11) : 1262～1267 ISSN 0379 - 4 172 P CRb a s e d S c r e e ni n g BA C Li b r a r y a n d Di r e c t En d S e q u e n ci n g of BA C Cl o n e s 1 ,2 2 , ① HE CongFen , Takao Komat suda ( 1 Key Lab of Plant Re source s Re se arch and Develop ment , Beijing Technology and Busine ss Univer sity , Beijing 　100037 , China ; 2 Genetic Diver sity Dep artment , National Institut e of Agrobiolo gical Science , Tsukuba 　305 8602 , J ap an) A b s t r a c t : A PCR ba se d strat egy wa s develop e d which re quire d four st ep s to identify po sitive BAC clone s from barley BAC (bact erial artificial chromo some ) library . In the protocol ,two level s of BAC DNA pool s ( sup erpool and pool) were prep are d for analy si s. One pool i s made of one plat e DNAs and one sup erpool i s made of mixing t en consecutive 1/ 100 dilut e d pool DNAs ( 1～10 ,11～20 ect) . Fir st ,sup erpool DNAs were analy se d and then 10 pool DNAs cont aine d in every po sitive sup erpool were analy se d . Once po sitive BAC plat e s were identifie d , the bact erial culture s were dipp e d into PCR mixture s and re action i s made to identify po sitive BAC clone s. The BAC clone s identifie d by e ach marker were group e d into contigs. BACend sequence wa s obt aine d from BACs within e ach contig and primer s were de signe d for the next st ep chromo some walking . In ca se of the BAC ends belong to rep etitive se quence ,the primer s were de signe d ba se d on the s