1. [Construction and preliminary identification of subtracted cDNA library of leukemia cell line K562]
- Author
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Yan-Bin, Song, Wen-Li, Ma, Chun-Qiong, Feng, Rong, Shi, Xiang-Ming, Mao, Bao, Zhang, and Wen-Ling, Zheng
- Subjects
DNA, Complementary ,Gene Expression Profiling ,Restriction Mapping ,Humans ,Nucleic Acid Hybridization ,RNA, Messenger ,Sequence Analysis, DNA ,Cloning, Molecular ,K562 Cells ,Polymerase Chain Reaction ,Gene Library - Abstract
Subtractive hybridization technology is a common method to screen and clone differentially expressed genes. This study was to construct subtracted cDNA library of leukemia cell line K562, and screen for differentially expressed genes.cDNA fragments of K562 cells (tester), prepared by restriction display (RD), were subtracted with the Sau3A I-digested cDNA fragments of normal lymphocytes (driver). The subtracted cDNA fragments were re-amplified, and cloned into pMD18-T vectors. Positive clones were selected by blue-white screening. The inserts in plasmid were amplified by polymerase chain reaction (PCR), and some of which were sequenced.The subtracted library contained 360 positive clones with cDNA fragments distributed mainly from 200 to 800 bp. The 50 randomly sequenced clones were derived from 42 known genes.Specific subtracted cDNA library of K562 cells was successfully constructed with reliable quality, and may be used to further screen and clone differentially expressed genes of K562 cells.
- Published
- 2005