Region-specific cosmids and STRPs identified by chromosome microdissection and FISH.
A strategy for identifying short tandem repeat (STR)-containing cosmid clones from a specific chromosomal region is described. The approach is based on the use of uncloned, PCR-amplified DNA derived from chromosome microdissection and pooled groups of STR sequences as hybridization probes to screen a cosmid library. Cosmid clones that display a positive signal common to both hybridizations are then characterized for repeat length polymorphisms. This method has been applied to chromosome bands 17q12-q21, a region that includes a gene (BRCA1) involved in early onset familial breast and ovarian cancer. Of 1536 chromosome 17-specific cosmid clones tested, 38 were identified by the dual screening procedure. Fluorescence in situ hybridization revealed that 19 cosmids originated from the microdissected target region. Thirteen of the 19 cosmids were mapped between markers flanking the BRCA1 region and selected for further characterization. Tetranucleotide repeats were identified in 10 of these 13 cosmids. Primers designed for each marker were tested on a panel of 80 CEPH parents for allele sizes, frequencies, and observed heterozygosities. From these studies six polymorphic and one nonpolymorphic STRs were identified. A similar approach should be applicable for screening whole genomic or chromosome-specific cosmid libraries in efforts to isolate new polymorphic markers from any chromosomal region of interest.
Flejter
W L
WL
Department of Pediatrics, University of Michigan, Ann Arbor 48109, USA.
Bennett-Baker
P
P
Barcroft
C L
CL
Kiousis
S
S
Chamberlain
J S
JS
D001483:Base Sequence; D001943:Breast Neoplasms / Q000235:genetics; D002874:Chromosome Mapping; D002886:Chromosomes, Human, Pair 17; D003360:Cosmids; D005260:Female; D006801:Humans; D017404:In Situ Hybridization, Fluorescence; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes; D010051:Ovarian Neoplasms / Q000235:genetics; D016133:Polymerase Chain Reaction; D011110:Polymorphism, Genetic; D012091:Repetitive Sequences, Nucleic Acid
D015345:Oligonucleotide Probes