遗传 ›› 2010, Vol. 32 ›› Issue (8): 824-828.doi: 10.3724/SP.J.1005.2010.00824

• 研究报告 • 上一篇    下一篇

染色体特异DNA文库的构建与鉴定

孙雷1, 刘新雄1, 陈哲1, 张明2, 陆阳清2, 卓永光1, 符可鹏1, 樊祖茜1   

  1. 1. 钦州市妇幼保健院, 钦州 535000; 2. 广西大学动物繁殖研究所, 南宁 530005
  • 收稿日期:2009-11-15 修回日期:2010-04-13 出版日期:2010-08-20 发布日期:2010-08-23
  • 通讯作者: 孙雷 E-mail:sunshijie12345@163.com
  • 基金资助:

    2009年钦州市科学研究与技术开发计划项目(合同编号:20092804)资助

Construction and identification of the chromosome specific DNA li-brary

SUN Lei1, LIU Xin-Xiong1, CHEN Zhe1, ZHANG Ming2, LU Yang-Qing2, ZHUO Yong-Guang1, FU Ke-Peng1, FAN Zu-Qian1   

  1. 1. Qinzhou Women and Children Healthcare Hospital, Qinzhou 535000, China; 2. Animal Reproduction Institute, Guangxi University, Nanning 530005, China
  • Received:2009-11-15 Revised:2010-04-13 Online:2010-08-20 Published:2010-08-23
  • Contact: SUN Lei E-mail:sunshijie12345@163.com

摘要: 为构建人类21号染色体特异DNA文库, 以应用于人类遗传疾病的鉴定和研究, 文章采用循环温度梯度法溶解释放微分离的人外周血细胞21号染色体DNA, 将其进行简并寡核苷酸引物PCR(Degenerate oligo nucleotide primer-PCR, DOP-PCR)扩增后, 利用100~500 bp和500~2 000 bp分段回收纯化的两种不同片段大小的DOP-PCR产物构建染色体特异DNA文库, 并分别采用荧光原位杂交(Florescence in situ hybridization, FISH)和斑点杂交对DOP-PCR产物的来源和随机取样的文库克隆进行检测以评估所构建DNA文库的特异性。结果表明: 循环温度梯度法能有效溶解释放微分离的21号染色体DNA; 通过对DOP-PCR产物的分段回收纯化和克隆, 增加了大片段DNA的连接效率; 利用FISH技术和斑点杂交双重鉴定实验证明了文库的特异性, 从而构建了21号染色体特异的DNA文库, 并建立了构建染色体特异DNA文库及检测其特异性的方法, 为21号染色相关遗传疾病的鉴定和研究奠定了基础。

关键词: 染色体微分离与微切割, DOP-PCR, 染色体特异DNA文库, 21号染色体

Abstract: The objective of the present study was to construct a human chromosome 21 specific DNA library for further use in research of genetic disease. Human chromosome 21 microdissected from the peripheral blood cells were subjected to repeatedly incubation in gradient temperature bath to release DNA. The library of chromosome 21 was constructed using the DNA fragment of 100–500 bp and 500–2 000 bp recovered from the products of DOP-PCR. Florescence in situ hy-bridization (FISH) and dot blotting analyses were carried out to assess the chromosome 21 specificity of the DNA library. The results indicated that DNA of chromosome 21 was released easily after repeatedly incubation in gradient temperature bath. Recovery of DNA fragments from DOP-PCR in different size ranges improved the efficiency of cloning of large fragments. Both FISH and dot blotting analyses revealed that the DNA library constructed in this study was chromosome 21-specific. This DNA library facilitates identification and investigation of the chromosome 21 related abnormality.

Key words: chromosome 21, chromosome microdissection, DOP-PCR, chromosome-specific DNA library