Cloning differentially expressed genes of human pancreatic cancer using suppressive subtractive hybridization
GE Wen-pin
Abstract：Objective To clone differentially expressed genes of human pancreatic cancer by suppressive subtractive hybridization(SSH). Methods Total RNA and mRNA were isolated from human pancreatic cancer(as tester) and normal paracancerous pancrease tissues(as driver), respectively. Then double-strand cDNA which was synthesized and restricted by rsa 1 enzyme Tester cDNA, was divided into two groups and ligated to the specific ada-ptor 1 and adaptor 2R, respectively. Tester cDNA was hybridized with driver cDNA twice and underwent neared PCR twice. Then the PCR products were cloned into T/A vector and transformed E. coli TOP 10F'. to set up the suppres-sive subtractive cDNA library. The positive clones randomly selected in the library were sequenced and analyzed for homology in the Gen Bank databases with BLAST. Results The amplified library contained approximately 251 white bacteria clones Random analysis of 53 white clones with PCR amplification showed that 50 clones contained inserted fragments, The positive rate is 96%. The size of inserted fragments was between 600-600bp. The 50 positive clones were selected for further sequencing and 45 usable sequences were obtained. After BLAST homology analysis,44 of them were found to be known genes,and 1 of them was found to be unknown gene with on functional clues,and may be a novel gene. Conclusions A suppressive cDNA library of human pancreatic cancer is constructed successfully by SSH. One novel differentially expressed gene is found with SSH and might be novel pancreatic cancer associated.
Keywords：Pancreatic neoplasmsSuppressive subtractive hybridizationGene libraryClone
Publication Date：2010-01-01
Online Publishing Date：2026-09-14(First online date of this platform, not the publication date of the document)