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

A TECHNIQUE OF CONSTRUCTING HUMAN CHROMOSOMAL BAND SPECIFIC PROBE POOLS USING DEGENERATE OLIGONUCLEOTIDE PRIMER

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（ｄｅｇｅｎｅｒａｔｅｏｌｉｇｏｎｕｃｌｅｏｔｉｅｄｐｒｉｍｅｒｅｄ－ＰＣＲ，ＤＯＰ－ＰＣＲ）扩增，并用荧光原位杂交（ｆｌｕｏｒｅｓｃｅｎｃｅｉｎｓｉｔｕｈｙｂｒｉｄｉｚａｔｉｏｎ，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。

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 1.2×10 4 clones. The average size of inserts was about 420bp by analysis of 30 positive clones. The rate of single copy and low repeated sequences was about 81%(178/220), while the rate of middle repeated and high repeated sequences was about 19%(42/220).Conclusion The results proved that the modified microdissection combining DOP PCR technique provided a simple and efficient method to construct the human chromosome band specific probe pools and might contribute to gene cloning and complete sequencing of human genome.