Catch-linker + PCR labeling: a simple method to generate fluorescence in situ hybridization probes from yeast artificial chromosomes.
A simple and efficient method to generate hapten-labeled DNA fragments from a trace amount of YAC DNA isolated by PFGE is described. After agarase digestion of the gel slice containing the resolved YAC recombinant, the purified DNA is digested with Sau3Al and a compatible CL oligonucleotide duplex ligated on. A probe is generated by PCR amplification using a primer complementary to the CL with a single biotin moiety incorporated at the 5' end. When used as a FISH probe, this material yields mapping results superior to Alu-PCR or whole YAC labeling methods and allows sensitive detection of chimerism.
Shibasaki
Y
Y
Medical Research Council (MRC) Human Genetics Unit, Western General Hospital, Edinburgh, Scotland, UK.
Maule
J C
JC
Devon
R S
RS
Slorach
E M
EM
Gosden
J R
JR
Porteous
D J
DJ
Brookes
A J
AJ
D001483:Base Sequence; D002678:Chimera; D018244:Chromosomes, Artificial, Yeast; D011088:DNA Ligases; D004271:DNA, Fungal / Q000235:genetics / Q000302:isolation & purification; D006801:Humans; D017404:In Situ Hybridization, Fluorescence / Q000379:methods; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D009838:Oligodeoxyribonucleotides; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity
D004271:DNA, Fungal; D009838:Oligodeoxyribonucleotides; D011088:DNA Ligases