Use of thymidine labelled vector (T-vector) for cloning and sequencing of enzymatically amplified ribosomal RNA genes of helicobacter pylori. | [Pakistan Journal of Medical Research • 1996]

Author(s):

1. Mubashir A. Khan: Pakistan Medical Research Council, Islamabad, Pakistan

2. Bohumil S. Drasar: Clinical Science Department, Bacterial Molecular Genetics, London School of Hygene & Tropical Medicine, London, U. K

3. Neil G. Stoker: Pakistan Medical Research Council, Islamabad, Pakistan; Clinical Science Department, Bacterial Molecular Genetics, London Sch. Hyg. & Trop. Medical, London, U. K

Abstract:

Part of the 16S RNA (rRNA) genes of Helicobacter pylori was amplified by using polymerase chain reaction. Amplified product was cloned into modified bacteriophage vector M13mp18 (called T vector). Nineteen potentially recombinant clones were obtained, out of which eleven have shown the definite presence of DNA of interest, and hence evidenced the successful cloning of amplified product. Single stranded DNA from these recombinant clones were extracted and used for sequencing. Sequencing of cloned DNA was performed in both orientations using M13 universal primer and other sequencing primers generally used for eubacterial species. Complete nucleotide sequence of the cloned fragment has shown 77% homology with the same region of 16S rRNA gene of Escherichia coli. The method used in this study is very general so that a single batch of prepared vector can be used for cloning of any PCR product. The method is also applicable for plasmid vectors.

Page(s): 161-166

DOI: DOI not available

Published: Journal: Pakistan Journal of Medical Research, Volume: 35, Issue: 4, Year: 1996

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