A quantitative method for analyzing specific DNA sequences directly from whole cells.
A quick, accurate assay for specific DNA sequences is described in which whole cells are treated with 0.4 M sodium hydroxide at 80 degrees C. DNA is relatively resistant to alkaline hydrolysis, whereas proteins and RNA are degraded rapidly. The DNA in NaOH is then transferred through a slot directly onto a nylon membrane and hybridized with a probe. Since the procedure is so simple, many samples can be analyzed in a short time. A single-copy gene can be detected in as few as 1000 cells and, since the DNA from 10(5) cells can be loaded through a single slot, the sensitivity is sufficient to detect one specific DNA sequence per 100 cells. Accurate quantitative analysis can be achieved by normalizing the amount of DNA available for hybridization in each slot, using a probe derived from total DNA.
McIntyre
P
P
Imperial Cancer Research Fund Laboratories, London, England.
Stark
G R
GR
D000818:Animals; D001483:Base Sequence; D002478:Cells, Cultured; D004247:DNA / Q000032:analysis* / Q000235:genetics; D015342:DNA Probes; D005784:Gene Amplification; D006358:Hot Temperature; D006868:Hydrolysis; D009693:Nucleic Acid Hybridization; D012972:Sodium Hydroxide
D015342:DNA Probes; D012972:Sodium Hydroxide; D004247:DNA