Mapping dispersed repetitive loci using semi-specific PCR cloning and somatic cell hybrid mapping

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 locat...

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Bibliographic Details
Published in:Nucleic acids research 2000-12, Vol.28 (23), p.E103-103
Main Authors: Deng, Y M, Lee, J H, Moran, C, Jin, J H, Tuch, B E, Rawlinson, W D
Format: Article
Language:English
Subjects:
Animals
Cell Line
chromosome 17
Chromosome Mapping
Cloning, Molecular
DNA - chemistry
DNA - genetics
Endogenous Retroviruses - genetics
Female
Humans
Male
Molecular Sequence Data
NAR Methods Online
Polymerase Chain Reaction
porcine endogenous retrovirus
Radiation Hybrid Mapping
Repetitive Sequences, Nucleic Acid - genetics
Sequence Analysis, DNA
Swine
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Summary: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.
ISSN:1362-4962
0305-1048
1362-4962
DOI:10.1093/nar/28.23.e103