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[A technique of constructing human chromosomal band-specific probe pools using degenerate oligonucleotide primer]
1State Key Laboratory of Medical Genetics of China, Hunan Medical University, Changsha, Hunan Province, 410078 P.R.China.
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 band 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 linke up. The DH5alpha 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 x 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.