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A mercury-thiol affinity system for rapid generation of overlapping labeled DNA fragments for DNA sequencing



A mercury-thiol affinity system for rapid generation of overlapping labeled DNA fragments for DNA sequencing



Nucleic Acids Research 10(13): 4009-4025



We describe an in vitro protocol for quickly generating overlapping terminal-labeled restriction fragments for DNA sequence analysis via the Maxam-Gilbert technique. The protocol involves introducing mercurated nucleotides into one end of a region to be sequenced, partial digestion with several restriction enzymes and terminal-labeling, separation of the mercurated restriction enzymes and terminal-labeling, separation of the mercurated restriction fragments from non-mercurated ones on a thiol column and resolution of the different mercurated fragments on one preparative agarose gel. The protocol was used to determine the nucleotide sequence of a 980 base pair cDNA that contains the coding region for a variable surface glycoprotein of Trypanosoma brucei. It could just as quickly and easily be used to obtain many terminal-labeled overlapping restriction fragments covering a region of several kilobases.

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Accession: 001038353

Download citation: RISBibTeXText

PMID: 7050914

DOI: 10.1093/nar/10.13.4009


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