[A method for determining DNA sequence by labeling the end of the molecule and cleaving at the base. Isolation of DNA fragments, end-labeling, cleavage, electrophoresis in polyacrylamide gel and analysis of results].
We elaborate basic chemical principles and current laboratory procedures for sequencing end-labeled DNA by partial cleavage and gel electrophoresis (A. M. Maxam and W. Gilbert, Proc. Natl. Acad. Sci. USA, 1977, v. 74, p. 560-564). We provide step-by-step protocols for 32P-labeling DNA ends, segregating the labeled ends by cutting with a second restriction enzyme or separating strands, partially cleaving the DNA at specific bases with reagents, electrophoresing the labeled products of cleavage on sequencing gels, and interpreting sequencing band patterns. Many of these procedures have been condensed, to make them faster and easier, and some are new. We also discuss sequencing strategies, and suggest a technique which will reduce plasmid or viral DNA to a collection of singly-end-labeled fragments in one day, for efficient sequencing of these chromosomes in 250-nucleotide blocks.
D001483:Base Sequence; D055598:Chemical Phenomena; D002621:Chemistry; D004247:DNA / Q000032:analysis*; D004591:Electrophoresis, Polyacrylamide Gel; D007553:Isotope Labeling; D008722:Methods
D004247:DNA
Metod opredeleniia posledovatel'nosti DNK (sekvenirovaniia) posle vvedeniia metki v konets molekuly i rasshchepleniia ee po osnovaniiam. Poluchenie fragmentov DNK, vvedenie kontsevoĭ metki, reaktsii rasshchepleniia, élektroforez v poliakrilamidnom gele i obrabotka rezul'tatov.