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AIM:The genomes of Helicobacter pylori (H.pylon) fromdifferent individuals are different.This project was toidentify the strain specific DNA sequences between twoclinical H.pylori isolates by suppression subtractivehybridization (SSH).METHODS:Two clinical H.pyloriisolates,one from gastriculcer (GU,tester) and the other from non-ulcer dyspepsia(NUD,driver),were cultured and the genomic DNA wasprepared and submitted to AluI digestion.Then two differentadaptors were ligated respectively to the 5’-end of twoaliquots of the tester DNA fragments and SSH was madebetween the tester and driver DNA.The un-hybridized testerDNA sequences were amplified by two sequential PCR andcloned into pGEM-T-Easy Vector.The tester strain specificinserts were screened and disease related DNA sequenceswere identified by dot blotting.RESULTS:Among the 240 colonies randomly chosen,50contained the tester strain specific DNA sequences.Twentythree inserts were sequenced and the sizes ranged from261 bp to 1 036 bp.Fifteen inserts belonged to the H.pyloriplasmid pHPO100 that is about 3.5 kb and codes a replicationprotein A.Other inserts had patches of homologous to thegenes of H.pyloriin GenBank.Various patterns of dot blotswere given and no GU strain unique DNA sequences werefound when 4 inserts were used as probes to screen thegenomic DNA from 27 clinical isolates,8 from GU,12 fromduodenum ulcer (DU),4 from GU-DU,2 from NUD and 1from gastric cancer (GC).But a 670 bp DNA fragment (GU198)that was a bit homologous to the 3“-end of the gene ofthymidylate kinase was positive in 7 GU strains (7/8),3 GU-DU strains (3/4) and 3 DU strains (3/12).A 384 bp fragment(GU79) of the replication gene A (repA) was positive only in4 H.pyloriisolates,2 from GU and 2 from GU-DU.CONCLUSION: Differences exist in the genes of different H.pylori isolates. SSH is very effective to screen H.pylori strain specific DNA sequences between two clinical isolates, and some of these sequences may have clinical significance.
AIM: The genomes of Helicobacter pylori (H. pylon) from different individuals are different. This project was to identify the strain specific DNA sequences between twoclinical H. pylori isolates by suppression subtractive hybridization (SSH). METHODS: Two clinical H. pyloriisolates, one from gastriculcer (GU, tester) and the other from non-ulcer dyspepsia (NUD, driver), were cultured and the genomic DNA wasprepared and submitted to AluI digestion.Then two different adapters were ligated respectively to the 5’-end of two aliquots of the tester DNA fragments and SSH was made between the tester and driver DNA. The un-hybridized tester DNA sequences were amplified by two sequential PCR and cloned into pGEM-T-Easy Vector. The tester strain specific in-serts were screened and disease related DNA sequences were identified by dot blotting .RESULTS: Among the 240 colonies randomly chosen, 50contained the tester strain specific DNA sequences. Twentythree inserts were sequenced and the sizes ranged from 261 bp to 1 036 bp.Fif teen inserts belonged to the H. pyloriplasmid pHPO100 that is about 3.5 kb and codes a replication protein A. Prior inserts had patches of homologous to the genes of H. pylori in GenBank. Variants patterns of dot blotswere given and no GU strains unique DNA sequences were found when 4 Insert were used as probes to screen the genomic DNA from 27 clinical isolates, 8 from GU, 12 from duodenum ulcer (DU), 4 from GU-DU, 2 from NUD and 1 from gastric cancer (GC) .But a 670 bp DNA fragment ) that was a bit homologous to the 3 ”" - end of the gene of thymidylate kinase was positive in 7 GU (7/8), 3 GU-DU (3/4) and 3 DU (3/12). A 384 bp fragment (GU79) of the replication gene A (repA) was positive only in4 H. pylori isolates, 2 from GU and 2 from GU-DU. CONCLUSION: Differences exist in the genes of different H. pylori isolates. SSH is very effective to screen H. pylori strain specific DNA sequences between two clinical isolates, and some of these sequences may have clinical significance.