Generation of chromosome paints: approach for increasing specificity and intensity of signals.
Chromosome painting is a widely used technique, and the two principal means of generating probes for such experiments involve DNA isolation by chromosome flow sorting and by chromosome microdissection. Frequently, chromosome paints are bright and specific; however, on occasion, signals can be weak and nonspecific, particularly for microdissected probes. Reasons for this have been attributed to co-amplification of non-target DNA and the formation of primer concatamers during degenerate oligonucleotide primed (DOP)-PCR. Here we describe a technique of circumventing this problem by sequence enrichment. It involves co-hybridization of DOP-PCR biotinylated microdissected material and linkered genomic DNA. Biotinylated DNA fragments captured on streptavidin-coated paramagnetic beads are eluted and amplified by PCR using a single primer complementary to the linker arm.
Masabanda
Julio S
JS
Department of Biological Sciences, Brunel University, Uxbridge, Middlesex, UB8 3PH, UK.
Griffin
Darren K
DK
D000818:Animals; D002645:Chickens; D020223:Chromosome Painting / Q000379:methods*; D033481:Chromosomes, Mammalian / Q000648:ultrastructure*; D006224:Cricetinae; D017404:In Situ Hybridization, Fluorescence; D008856:Microscopy, Fluorescence / Q000379:methods; D016133:Polymerase Chain Reaction / Q000379:methods; D011786:Quality Control; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity