Discriminatory 32P 3'-end labeling of restriction endonuclease co-digested DNA fragments.
We present an improved method for selectively labeling specific DNA fragments in a mixture of restriction fragments. lambda DNA, used to develop the procedure, was digested with a combination of restriction enzymes and treated, at various incubation temperatures, with reverse transcriptase and one or more [alpha-32P]dNTP molecules. The labeled fragments were subjected to agarose gel electrophoresis and detected by autoradiography. We were able to direct the 3'-end labeling to precise subpopulations of DNA fragments, generated by multiple restriction enzyme digestions. We also show that labeling selectivity of DNA restriction fragments, by reverse transcriptase, is affected by the incubation temperature used during the labeling reaction. This paper describes an approach to 3'-end label select DNA fragments with reverse transcriptase and [alpha-32P]dNTPs. The procedure permits the bypassing of time-consuming isolation and purification steps required, by conventional radiolabeling techniques, to radiolabel specific DNA fragments.
LeBlond
G F
GF
Division of Biophysics, School of Hygiene and Public Health, Johns Hopkins University, Baltimore, Maryland 21205.
Ts'o
P O
PO
D001345:Autoradiography / Q000379:methods; D004247:DNA; D004262:DNA Restriction Enzymes; D004279:DNA, Viral; D004587:Electrophoresis, Agar Gel / Q000379:methods; D005796:Genes; D010761:Phosphorus Radioisotopes; D011865:Radioisotope Dilution Technique; D015183:Restriction Mapping
D004279:DNA, Viral; D010761:Phosphorus Radioisotopes; D004247:DNA; D004262:DNA Restriction Enzymes