Generation of chromosome fragment specific bovine DNA sequences by microdissection and DOP-PCR.
A rapid procedure for the defined isolation and characterization of single bovine chromosome fragment specific probes is described. This has been developed as a technical prerequisite for the directed generation of bovine DNA sequences. The specific regions 1q13-24, 5q21-24, 6q31-32, 7q21-22, 12q24-ter, and 20q12-ter of bovine GTG-banded metaphase chromosomes were microdissected and amplified by PCR with a degenerate oligonucleotide primer and subsequently cloned into pBluescript II SK. The DNA probes generated were characterized by gel electrophoresis, dot blot analysis and rehybridization in situ to GTG-banded metaphase spreads. The position and size of the hybridization sites on the chromosomes correspond exactly to the dissected chromosome areas and indicate the complexity and specificity of the microdissected and amplified chromosome material.
Goldammer
T
T
Forschungsbereich Molekularbiologie, Forschungsinstitut für die Biologie landwirtschaftlicher Nutztiere, D 18196 Dummerstorf, Germany.
Weikard
R
R
Brunner
R M
RM
Schwerin
M
M
D000818:Animals; D001483:Base Sequence; D002417:Cattle / Q000235:genetics*; D002871:Chromosome Banding; D017931:DNA Primers / Q000235:genetics; D015342:DNA Probes / Q000235:genetics* / Q000302:isolation & purification*; D004210:Dissection / Q000379:methods; D017404:In Situ Hybridization, Fluorescence; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*
D017931:DNA Primers; D015342:DNA Probes