Nucleic Acids Research, 2000, Vol. 28, No. 23 e103
© 2000 Oxford University Press
Mapping dispersed repetitive loci using semi-specific PCR cloning and somatic cell hybrid mapping
1Virology Division, Department of Microbiology, SEALS and 2Pancreas Transplant Unit, Department of Endocrinology, Prince of Wales Hospital, Randwick, NSW 2031, Australia, 3Department of Animal Science, University of Sydney, NSW 2006, Australia, 4Department of Medicine and 5School of Pathology, Faculty of Medicine, The University of New South Wales, Sydney 2052, Australia and 6Beijing Institute of Geriatrics, Beijing Hospital, Beijing, P.R. China
A simple and effective method based upon semi-specific PCR followed by cloning has been developed. Chromosomal mapping of the generated fragment on a somatic cell hybrid panel identifies the chromosomal position, and yields a unique sequence tag for the site. Using this method, the chromosomal location of one porcine endogenous retrovirus (PERV) was determined. The porcine genomic sequences were first amplified by PCR using a PERV-specific primer and a porcine short interspersed nuclear element (SINE)-specific primer. PCR products were cloned, and those sequences that contained PERV plus flanking regions were selected using a second round of PCR and cloning. Sequences flanking the PERV were determined and a PERV-B was physically mapped on porcine chromosome 17 using a somatic hybrid panel. The general utility of the method was subsequently demonstrated by locating PERVs in the genome of PERV infected human 293 cells. This method obviates the need for individual library construction or linker/adaptor ligation, and can be used to quickly locate individual sites of moderately repeated, dispersed DNA sequences in any genome.
* To whom correspondence should be addressed at: Virology Division, Department of Microbiology, SEALS, Prince of Wales Hospital, High Street, Randwick, NSW 2031, Australia. Tel: +61 2 93829113; Fax: +61 2 93984275; Email: w.rawlinson@unsw.edu.au