Selective digestion of mouse chromosomes with restriction endonucleases. Oligonucleotide priming of single-stranded DNA produced with exonuclease III.
L-929 mouse chromosomes prepared for electron microscopy have been treated with MspI, EcoRI, and HaeIII restriction endonucleases (REs). RE-induced nicks were amplified with exonuclease III to obtain single-stranded DNA (ss-DNA) motifs. The ss-DNA produced was enough to permit hybridization of a series of random oligonucleotides. These can be used as primers, which are extended by the Klenow fragment using non-isotopic labelled dUTP. The incorporation of biotinylated dUTP is detected with a gold-tagged streptoavidin as the reporter molecule. This allows, in mouse chromosomes, the detection of different rates of sensitivity to the digestion with specific REs in distinct intraheterochromatic DNA subsets. In addition, these results show that enzymatic production of ss-DNA seems to be adequate for electron microscopy work since the chromatin fiber is preserved better than in denatured DNA produced with heat, NaOH, or formamide.
Departamento de Biología, Facultad de Ciencias, Universidad Autónoma de Madrid, Spain.
J
J
Gosálvez
C
C
López-Fernández
C
C
García de la Vega
R
R
Mezzanotte
J L
JL
Fernández
V
V
Goyanes
D000818:Animals; D002478:Cells, Cultured; D002875:Chromosomes / Q000378:metabolism / Q000648:ultrastructure*; D004262:DNA Restriction Enzymes / Q000378:metabolism*; D004277:DNA, Single-Stranded / Q000378:metabolism; D005090:Exodeoxyribonucleases / Q000378:metabolism*; D051379:Mice; D008854:Microscopy, Electron / Q000379:methods; D009841:Oligonucleotides / Q000378:metabolism; D015183:Restriction Mapping
D004277:DNA, Single-Stranded; D009841:Oligonucleotides; D005090:Exodeoxyribonucleases; C020133:exodeoxyribonuclease III; D004262:DNA Restriction Enzymes