[Construction and preliminary identification of subtracted cDNA library of leukemia cell line K562].
OBJECTIVE
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.


METHODS
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.


RESULTS
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.


CONCLUSIONS
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.
Song
Yan-Bin
YB
Institute of Molecular Biology, South Medical University, Guangzhou, Guangdong, 510515, P.R.China.
Ma
Wen-Li
WL
Feng
Chun-Qiong
CQ
Shi
Rong
R
Mao
Xiang-Ming
XM
Zhang
Bao
B
Zheng
Wen-Ling
WL
D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics*; D020869:Gene Expression Profiling; D015723:Gene Library; D006801:Humans; D020014:K562 Cells; D009693:Nucleic Acid Hybridization / Q000379:methods; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000235:genetics; D015183:Restriction Mapping / Q000379:methods; D017422:Sequence Analysis, DNA
D018076:DNA, Complementary; D012333:RNA, Messenger