[Cloning associated genes using microdissection-cDNA PCR-SSH in gastric dysplasia]

Abstract

Objective: To construct cDNA subtracted libraries from gastric dysplasia and further screen differentially expressed genes.

Methods: Relatively pure dysplasia and normal tissue were procured by manual microdissection, and amplified by cDNA-PCR, which was used to carry on for suppression subtractive hybridization (SSH). Subtracted cDNA fragments were linked with vector, cloned, screened, sequenced, and made homologous search. Differentially expressed fragments were verified by dot hybridization.

Results: Two subtracted cDNA libraries were constructed. Among 26 sequenced clones, 15 fragments corresponded to known genes, 3 fragments were known EST and 8 fragments were unknown EST (GenBank BQ164614-BQ164616, BQ291516-BQ291520). Fifteen fragments were verified to be differentially expressed in gastric dysplasia.

Conclusions: Subtracted cDNA libraries from gastric dysplasia are constructed using combination of microdissection-cDNA PCR and SSH setup in our laboratory. Some fragments have been screened and verified to help to search for novel associated genes with gastric carcinogenesis.