Rapid identification of polymorphic CA-repeats in YAC clones.
Positional cloning of rare disease genes depends on the availability of highly polymorphic markers near the disease loci. The most abundant class of polymorphic markers in the human genome is CA-repeats. We have developed a strategy for the rapid isolation of highly polymorphic CA-repeats from YAC clones. Total DNA of yeast clones containing partly overlapping YACs is digested with frequent cutter restriction enzymes, blotted and hybridized with a poly(CA/GT) probe under high stringency conditions that enable preferential detection of long CA-repeats. The repeats detected in this way are isolated by PCR using vectorette linkers, sequenced, and appropriate flanking markers are constructed for genotyping. All of the CA-repeats identified using this approach were highly polymorphic. This simple and rapid approach should allow the development of highly polymorphic markers at any genomic region cloned in YACs.
Rotman
G
G
Department of Human Genetics, Sackler School of Medicine, Tel Aviv University, Ramat Aviv, Israel.
Vanagaite
L
L
Collins
F S
FS
Shiloh
Y
Y
D015139:Blotting, Southern / Q000379:methods*; D018244:Chromosomes, Artificial, Yeast / Q000235:genetics*; D004262:DNA Restriction Enzymes / Q000737:chemistry; D004276:DNA, Satellite / Q000302:isolation & purification*; D005819:Genetic Markers; D016681:Genome, Fungal; D015894:Genome, Human; D006801:Humans; D011089:Polydeoxyribonucleotides; D016133:Polymerase Chain Reaction; D012091:Repetitive Sequences, Nucleic Acid; D017421:Sequence Analysis
D004276:DNA, Satellite; D005819:Genetic Markers; D011089:Polydeoxyribonucleotides; C044165:poly(d(G-T).d(C-A)); D004262:DNA Restriction Enzymes