Optimization of in situ hybridization to human metaphase chromosomes.
A simplified method describing optimal conditions for in situ hybridization to human chromosomes is presented. A 1.5-kb DNA fragment coding for part of the 28 S rRNA was subcloned into pSP65. Tritium-labeled RNA was synthesized as runoff transcripts and 39-67% of the labeled probes specifically hybridized to the nucleolar organizer regions of the acrocentric chromosomes. Denaturation performed with 70% formamide, 1 mM EDTA, 2 X SSC gave a high specific hybridization with both fresh chromosome spreads (1-8 weeks) and older preparations (3-6 months). To obtain good chromosome morphology and a high specific hybridization it was important to neutralize the final formamide denaturation mixture containing 70% formamide, 1 mM EDTA, and 2 X SSC, whereas it was unimportant to deionize the formamide. Freshly made slides denatured with 0.15 M NaOH in 70% ethanol hybridized equally well with the rRNA probe. Despite treatment of the chromosomes with RNases before denaturation the following proteinase K and the acetylation steps recommended could be omitted without degradation of the rRNA probe as judged from the high specific hybridization to the nucleolar organizer regions.
Lomholt
B
B
Institute of Medical Genetics, Panum Institute, Copenhagen N, Denmark.
Sørensen
P D
PD
Simonsen
H
H
Frederiksen
S
S
D000818:Animals; D002875:Chromosomes / Q000502:physiology*; D005559:Formamides; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008214:Lymphocytes / Q000502:physiology; D008677:Metaphase; D051379:Mice; D009693:Nucleic Acid Hybridization
D005559:Formamides; C031066:formamide