Construction of a Subtractive cDNA Library of the Age-related Cataract by an Improved Subtractive Hybridization and Selective PCR

In order to obtain an efficient subtractive cDNA library with larger fragments of age related cataract, an improved subtractive hybridization with biotin labeling and magnetic bead isolation to obtain the subtractive cDNA was used. The large fragments of the subtractive cDNA were amplified by using selective PCR method and directly inserted into T/A cloning vector and transformed into E.coli . The library contained 4 000 clones. 22 clones were selected at random and identified by the restriction digestion, 7 fragments were ≥1 000 bp, being 31 8% of the total, and 15 fragments were ≥750 bp, being 68 2% of the total. The 15 clones with fragments ≥750 bp were hybridized by reverse dot blotting hybridization to remove the false positive fragments. The positive clones were sequenced and compared with GenBank. 6 cDNA fragments of known genes and a novel gene were obtained. 4 of the 6 known genes were full length gene. The cDNA fragments are larger and the library is efficient. A high qualitative subtractive cDNA library with larger fragments can be constructed rapidly and effectively by improved subtractive hybridization and selective PCR method. This lays a foundation for further screening and identifying the specific expressed gene of age related cataract.