Isolation of region-specific probes from pig chromosome 6 by coincidence cloning.
Coincidence cloning is a technique that permits the isolation of sequences common to two independent sources of complex DNA, and this method has been used to isolate a set of probes from a region of porcine Chromosome (Chr) 6 containing the loci for glucosephosphate isomerase (GPI) and the skeletal muscle calcium release channel (CRC). Porcine DNA was specifically PCR-amplified from a pig x hamster hybrid cell line containing the centromere region (p1.2-q1.2) of pig Chr 6 and other pig chromosome fragments by use of a porcine SINE specific primer with an EcoRI site in the 5'-end. Flow-sorted Chr 6 preparations were amplified with the same SINE primer, but with a SalI site in the 5'-end. The products were digested with EcoRI and SalI respectively, combined, denatured, and reannealed. The heteroduplex molecules, containing both an EcoRI and a SalI cohesive end, were selected by cloning in SalI/EcoRI-digested pUC13. Approximately 40% of the primary clones contained a single SalI/EcoRI-insert, indicating that they are coincidence clones. The average insert size was 1.4 kb. Fluorescence in situ hybridization of a pool of 34 coincidence clones to pig chromosomes showed a preferential labeling of the centromere region and of the q2.5-q2.7 region of pig Chr 6. Nineteen coincidence clones were hybridized to SINE-PCR products from flow-sorted pig Chr 6 and to pig x rodent hybrid cell lines. Eighteen clones gave positive signals correlated with the GPI/CRC content of the source DNAs.
Frengen
E
E
Department of Biochemistry, Physiology and Nutrition, Norwegian College of Veterinary Medicine, Oslo.
Thomsen
P D
PD
Schmitz
A
A
Frelat
G
G
Davies
W
W
D000818:Animals; D001483:Base Sequence; D015220:Calcium Channels / Q000235:genetics; D002874:Chromosome Mapping; D003001:Cloning, Molecular; D006224:Cricetinae; D017931:DNA Primers / Q000235:genetics; D015342:DNA Probes / Q000235:genetics* / Q000302:isolation & purification; D005956:Glucose-6-Phosphate Isomerase / Q000235:genetics; D006822:Hybrid Cells; D017404:In Situ Hybridization, Fluorescence; D015336:Molecular Probe Techniques; D008969:Molecular Sequence Data; D018482:Muscle, Skeletal / Q000378:metabolism; D013552:Swine / Q000235:genetics*
D015220:Calcium Channels; D017931:DNA Primers; D015342:DNA Probes; D005956:Glucose-6-Phosphate Isomerase