Construction of subtracted cDNA library by suppression subtractive hybridization for differentially expressed genes in eosinophils from asthma patients.
- Author:
Jin-song XU
1
;
Shao-xi CAI
;
Fei ZOU
;
Wan-cheng TONG
;
Wei-ren WAN
;
Hai-jin ZHAO
Author Information
- Publication Type:Journal Article
- MeSH: Asthma; blood; genetics; DNA, Complementary; genetics; Eosinophils; metabolism; Gene Expression Profiling; Gene Expression Regulation; genetics; Gene Library; Humans; Male; Middle Aged; Nucleic Acid Hybridization
- From: Journal of Southern Medical University 2006;26(1):82-85
- CountryChina
- Language:Chinese
-
Abstract:
OBJECTIVETo construct a subtracted cDNA library of differentially expressed genes in eosinophils from asthma patients.
METHODSSuppression subtractive hybridization (SSH) was used to isolate the cDNA fragments of differentially expressed genes in the eosinophils of asthma patients before and after treatment. The cDNA fragments were directly inserted into T/A cloning vector to establish the subtractive library, followed by amplification of the library through E. coli transformation with calcium chloride and screening of blue and white clones of the transformants. One hundred positive bacterial clones were randomly picked and identified by colony PCR.
RESULTSThe amplified library contained more than 3,000 positive bacterial clones. Analysis of the randomly selected 100 white clones by PCR showed that 90% of the clones contained 100-500 bp inserts, which might be the cDNA fragments of differentially expressed genes in eosinophils of asthma patients before treatment.
CONCLUSIONA subtracted cDNA library of differentially expressed genes in the eosinophils of asthma patients before and after treatment is constructed successfully by SSH and T/A cloning techniques, which lays a solid foundation for screening and cloning new specific differentially.expressed genes in the eosinophils of asthma patients.
