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 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.
Deng
Y M
YM
Virology Division, Department of Microbiology, SEALS and Pancreas Transplant Unit, Prince of Wales Hospital, Randwick, NSW 2031, Australia.
Lee
J H
JH
Moran
C
C
Jin
J H
JH
Tuch
B E
BE
Rawlinson
W D
WD
D000818:Animals; D002460:Cell Line; D002874:Chromosome Mapping; D003001:Cloning, Molecular; D004247:DNA / Q000737:chemistry / Q000235:genetics; D020077:Endogenous Retroviruses / Q000235:genetics; D005260:Female; D006801:Humans; D008297:Male; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D021101:Radiation Hybrid Mapping; D012091:Repetitive Sequences, Nucleic Acid / Q000235:genetics*; D017422:Sequence Analysis, DNA; D013552:Swine
D004247:DNA