PCR技术在快速筛选DNA文库中的应用  

Application of PCR in quickly screening DNA library

作　　者：赵永祥[1] 刘斌[1] 张昊宇[1] 胡冬煦[2] 杨进福[2] 夏家辉[3] 潘乾[3] 薛志刚[3] 

机构地区：[1]中南大学湘雅三医院心胸外科 [2]中南大学湘雅二医院心胸外科,湖南长沙410011 [3]中国医学遗传学国家重点实验室,湖南长沙410078

出　　处：《中国医学工程》2004年第4期48-51,共4页China Medical Engineering

基　　金：国家自然科学基金NO:30000203;国家211工程部省共建重点学科重大项目基金No[1998]6号;卫生部科学基金[No98-1-114];美国中华医学基金会(CMB)

摘　　要：目的探讨运用PCR (polymerase chain reaction)方法快速筛选DNA文库的可行性。方法以猪α-1,3GT cDNA片段为探针,用cDNA上的特异性序列合成的引物,采用噬菌斑原位杂交和PCR相结合的方法,对猪的gDNA文库进行α-1,3GT基因筛选,经酶切、Southern Blot、测序和荧光原位杂交定位。结果经过两次杂交和一次PCR即得信号很强的7个阳性单克隆,插入子均在8 kb以上,且包含第三内含子,其中3个片段测序证实含第三、第四外显子;荧光原位杂交证实该基因位于染色体1q2.10-q2.11。结论PCR可以应用于快速筛选DNA文库。Objective: To study the feasibility of PCR in screening DNA library. Methods: Porcineα-1,3GT gDNA fragment out of porcine gDNA library was screened by using α-1, 3GT cDNA probe hybridization and PCR with the specific primers on cDNA, and verified by enzymic digestion, southern blot and sequencing. Meanwhile, fluorescence in situ hybridization (FISH) was performed. Results: After screening α-1,3GT gDNA library with cDNA probe twice and PCR once, 7 positive clones with more than 8 kb length were obtained, including the third intron, 3 clones of them contain the third and fourth exons, which was mapped to porcine chromose 1q2.10-1q2.11. Conclusion: PCR is a reliable and quick technique to screen DNA library when it is used with probe hybridization together.

关 键 词：PCR DNA文库 猪 噬茵斑原位杂交 基因筛选 荧光原位杂交 

分 类 号：R394[医药卫生—医学遗传学]