Oligonucleotide-primed in situ DNA synthesis (PRINS): a method for chromosome mapping, banding, and investigation of sequence organization.
Oligonucleotides were annealed to complementary sequences in fixed human metaphase chromosomes and extended with DNA polymerase. The newly synthesized fragments were labeled by incorporating bio-11-dUTP instead of TTP, and the sites of synthesis were detected by immunocytochemistry, using fluorochromes as the reporter molecules. We have obtained clear localization with oligonucleotides from alphoid (centromeric sequences), simple sequence (satellite) DNAs, a variety of Alu-dispersed repeated sequences, and oligonucleotides derived from the Tetrahymena and Trypanosoma telomere-specific sequences. The simple sequence and alphoid oligonucleotides gave results at least comparable to those obtained using the whole molecule as a probe for in situ hybridization, whereas the Alu oligonucleotides produced a diversity of results which depended on the absolute length and location of the oligonucleotide within the Alu sequence. The telomere-specific oligomers also produced a variety of results. The G-rich Trypanosoma oligomer and its complementary C-rich sequence produced strong telomeric signals and some interstitial signals on mouse chromosomes, but only weak telomeric signals on human chromosomes. The G-rich Tetrahymena oligomer produced detectable telomeric signals on human chromosomes. The technique appears to be a valuable extension of present tools for mapping and examining the organization of DNA sequences within chromosomes.
MRC Human Genetics Unit, Western General Hospital, Edinburgh, Scotland, UK.
J
J
Gosden
D
D
Hanratty
J
J
Starling
J
J
Fantes
A
A
Mitchell
D
D
Porteous
D000818:Animals; D001483:Base Sequence; D002871:Chromosome Banding / Q000379:methods*; D002874:Chromosome Mapping / Q000379:methods*; D004247:DNA / Q000235:genetics*; D004261:DNA Replication / Q000187:drug effects*; D016650:Fluorescein-5-isothiocyanate; D006801:Humans; D007621:Karyotyping; D051379:Mice; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides / Q000494:pharmacology*; D014345:Trypanosoma / Q000235:genetics
D009838:Oligodeoxyribonucleotides; D004247:DNA; D016650:Fluorescein-5-isothiocyanate