黑麦B染色体显微切割和微克隆

MICRODISSECTION AND MICROCLONING OF THE B CHROMOSOME OF RYE ( Secale cereale )

显微分离出两条黑麦Ｂ染色体，经蛋白酶Ｋ处理，Ｓａｕ３ＡＩ酶切后，用ＬＡ－ＰＣＲ（ＬｉｎｋｅｒＡｄａｐｔｅｒＰＣＲ）方法进行扩增，得到０．２～２ｋｂ的ＤＮＡ片段．Ｓｏｕｔｈｅｒｎ杂交分析结果表明，此扩增产物来源于黑麦Ｂ染色体．将部分ＰＣＲ产物克隆到ｐＵＣ１９载体中，获得了５０００个克隆．

Two B chromosomes isolated from a metaphase cell of rye by microdissection, were digested with proteinase K and Sau3AⅠ, then the B chromosome DNA was amplified by LA PCR(Linker Adapter PCR).The PCR products are a smear ranged from 0.2 kb to 2 kb. The Southern hybridization showed that the PCR products were derived from the B chromosome of rye. Part of these PCR products were cloned into pUC19 and we obtained 5000 clones. This microdissection and microcloning method is a simple and general approach for constructing B chromosome DNA library.