Submitted to: Animal Genetics International Conference Proceedings
Publication Type: Abstract Only
Publication Acceptance Date: June 21, 1996
Publication Date: N/A
Technical Abstract:
Chromosomal microdissection has emerged as a powerful approach for obtaining DNA probes from specific chromosomal regions, thus facilitating construction of high-resolution physical and genetic maps. Dissection of chromosomes coupled with the use of the polymerase chain reaction enhances the amount of DNA available for cloning. We have successfully removed sub- -natural or artificial terminus. GTG banded porcine metaphase chromosomes were prepared using standard techniques except that care was taken to minimize the number of fixative steps to preclude extensive depurination. Chromosomal DNA was microdissected from targeted regions (13q33-q36; 1p21-p fine tip. Each dissected region was collected in a 0.5-ml Eppendorf tube containing 1 microliter of a solution comprised of 5mM EDTA (pH 8.0), 1 mg\ml proteinase K, and 30% PEG 6000. The "DOP-Shuttle-PCR" protocol developed by Yokoyama and Sakuragawa was employed, and amplified DNA was cloned into a T-tailed vector. Subgenomic libraries were screened for (CA)n repeats following protocols previously described. Eight fragments hybridizing to a (CA)10 probe have been sequenced. In four of these, the microsatellite was too close to the vector cloning site to develop primers on both sides. Primers from the other four positive clones have been developed and will be genotyped.
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