Patterns of digestion of human chromosomes by restriction endonucleases demonstrated by in situ nick translation.
A restriction enzyme-nick translation procedure has been developed for localizing sites of restriction endonuclease action on chromosomes. This method involves digestion of fixed chromosome preparations with a restriction enzyme, nick translation with DNA polymerase I in the presence of biotinylated-dUTP, detection of the incorporated biotin label with streptavidinalkaline phosphatase, and finally staining for alkaline phosphatase. Results obtained obtained on human chromosomes using a wide variety of restriction enzymes are described, and compared with results of Giemsa and Feulgen staining after restriction enzyme digestion. Results of nick translation are not in general the opposite of those obtained with Giemsa staining, as might have been expected. Although the nick translation procedure is believed to give a more accurate picture of the distribution of restriction enzyme recognition sites on chromosomes than Giemsa staining, it is clear that the results of the nick translation experiments are affected by accessibility to the enzymes of the chromosomal DNA, as well as by the extractability of the DNA.
Sumner
A T
AT
MRC Human Genetics Unit, Western General Hospital, Edinburgh, Scotland, UK.
Taggart
M H
MH
Mezzanotte
R
R
Ferrucci
L
L
D000469:Alkaline Phosphatase; D001426:Bacterial Proteins; D001482:Base Composition; D001483:Base Sequence; D002875:Chromosomes / Q000187:drug effects* / Q000378:metabolism; D004247:DNA / Q000032:analysis / Q000187:drug effects / Q000235:genetics; D004256:DNA Polymerase I / Q000494:pharmacology; D004262:DNA Restriction Enzymes / Q000494:pharmacology*; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007621:Karyotyping / Q000379:methods; D009711:Nucleotides / Q000032:analysis / Q000235:genetics; D014176:Protein Biosynthesis; D019809:Streptavidin
D001426:Bacterial Proteins; D009711:Nucleotides; D004247:DNA; D019809:Streptavidin; D004256:DNA Polymerase I; D004262:DNA Restriction Enzymes; D000469:Alkaline Phosphatase