您现在的位置： 绿色健康网 >> 医学论文 >> 基础医学论文 >> 正文 　

用简并寡核苷酸引物构建人类染色体区带特异性探针池的技术

邓昊　王文　夏家辉

　　【摘要】　目的　建立快速有效构建人类染色体区带特异性探针池及其文库的技术。方法　显微切割人类染色体3p23-p26，3q21-q22，4p12-p16区带，用简并寡核苷酸引物-PCR(degenerate oligonucleotied primered-PCR,DOP-PCR)扩增，并用荧光原位杂交(fluorescence in situ hybridization, FISH)检测扩增产物来源，再将区带特异性扩增产物克隆于pUC19载体构建区带特异性文库，以及用α-32PATP标记的gDNA行菌落原位杂交。结果　经FISH证实，3p23-p26，3q21-q22，4p12-p16探针池均在切割相应区带出现特异性信号。其中3q21-q22获得的1.2×104个阳性克隆。随机分析30个含插入片段的白色菌落，其插入片段平均约420bp。220个白色克隆菌落原位杂交示单拷贝和低度重复序列占81%(178/220)。　结论　改进的显微切割结合DOP-PCR为人类染色体探针池及文库构建建立了一个简易、高效的方法，这将有助于基因克隆和人类基因的全序列分析。
　　【关键词】　显微切割　　染色体区带　　简并寡核苷酸引物-聚合酶链反应　　探针池

A TECHNIQUE OF CONSTRUCTING HUMAN CHROMOSOMAL BAND-SPECIFIC PROBE POOLS USING DEGENERATE OLIGONUCLEOTIDE PRIMER　Deng Hao, Wang Wen, Xia Jiahui*.* State Key Laboratory of Medical Genetics of China, Hunan Medical University, Changsha 410078 P.R.China
　　【Abstract】　Objective　To establish a rapid and efficient technique of constructing human chromosomal band specific probe pools and their libraries.　Methods　A modified method of combining chromosome microdissection with degenerate oligonucleotide primed PCR (DOP-PCR) was used. 3p23-p26,3q21-q22 and 4p12-p16 bands from human chromosomes were microdissected and amplified as probe pools. The origins of the PCR products were determined by chromosome fluorescence in situ hybridization. The PCR products and pUC19 were digested by Xho　Ⅰ and Sal Ⅰ respectively, and linked up. The DH5α were transformed by the recombinated vectors as the specific band libraries. The inserts were digested by EcoR Ⅰ and Hind Ⅲ, then measured by electrophoretic analysis. And the copies of inserts were identified by in situ bacterial colony hybridization with genomic DNA.　Results　All the three probe pools showed the special yellow-green signals in their microdissection responsible bands. The sizes of DOP-PCR products ranged from 300bp to 1800bp. 3q21-q22 probe pool generated about 1.2×104 clones. The average size of inserts was about 420bp by analysi

[1] [2] [3] 下一页


  • 上一个医学论文：

  • 下一个医学论文：
    • 相关文章
    Copyright © 2006-2012 绿色健康网(www.gio.org.cn) All Rights Reserved.
    声明：网站信息仅供参考，不能作为诊断及医疗的依据。