Stability of randomly amplified polymorphic DNA fingerprinting in genotyping clinical isolates of Helicobacter pylori
H pylori genomes are highly diversified. This project was designed to genotype H pylori isolates by the polymerase chain reaction (PCR)-based randomly amplified polymorphic DNA (RAPD) fingerprinting technique and to verify its stability by Southern blotting and DNA sequencing. Clinical isolates of H...
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Published in | World journal of gastroenterology : WJG Vol. 9; no. 9; pp. 2021 - 2024 |
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Main Authors | , , |
Format | Journal Article |
Language | English |
Published |
United States
Department of Microbiology, Faculty of Medicine, National University of Singapore,5 Science Drive 2, Singapore 117595, Republic of Singapore
01.09.2003
Baishideng Publishing Group Inc |
Subjects | |
Online Access | Get full text |
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Summary: | H pylori genomes are highly diversified. This project was designed to genotype H pylori isolates by the polymerase chain reaction (PCR)-based randomly amplified polymorphic DNA (RAPD) fingerprinting technique and to verify its stability by Southern blotting and DNA sequencing.
Clinical isolates of H pylori were cultured from gastric antra and cardia of 73 individuals, and genomic DNA was prepared for each isolate. RAPD was carried out under optimized conditions. 23S rDNA was regarded as an internal control, and a 361 bp rDNA fragment (RDF) was used as a probe to screen the RAPD products by Southern blotting. Ten RDFs from different clinical isolates and the flanking regions (both upstream and downstream) of four RDFs were amplified and sequenced.
H pylori isolates from different individuals had different RAPD profiles, but the profiles for isolates cultured from different gastric sites of a given individual were identical in all but one case. Isolates from 27 individuals were RDF positive by Southern blotting. Sequences of the RDFs and their flanking regions were almost the same between the RDF positive and negative isolates as determined by Southern blotting. There was no binding site for random PCR primer inside the sequences.
RAPD is very useful in genotyping H pylori grossly on a large scale. However, it seems unstable in amplification of low yield fragments, especially those that do not appear as visible bands on the agarose gel stained with EB, since the primer is partially matched to the template. |
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Bibliography: | ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23 Correspondence to: Feng-Chan Han, Institute of Genetic Diagnosis, Fourth Military Medical University, 169 Changle West Road, Xi’an710033, Shaanxi Province, China. biohanfc@hotmail.com Telephone: +86-29-3374772 Fax: +86-29-3285729 Author contributions: All authors contributed equally to the work. |
ISSN: | 1007-9327 2219-2840 |
DOI: | 10.3748/wjg.v9.i9.2021 |