A mercury-thiol affinity system for rapid generation of overlapping labeled DNA fragments for DNA sequencing.
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.
D000818:Animals; D001483:Base Sequence; D002846:Chromatography, Affinity / Q000379:methods; D004247:DNA / Q000302:isolation & purification*; D003854:Deoxyribonucleotides; D004926:Escherichia coli / Q000235:genetics; D006023:Glycoproteins / Q000235:genetics; D008623:Mercaptoethanol; D008628:Mercury; D009838:Oligodeoxyribonucleotides / Q000302:isolation & purification*; D009841:Oligonucleotides / Q000302:isolation & purification*; D010957:Plasmids; D014346:Trypanosoma brucei brucei / Q000235:genetics
D003854:Deoxyribonucleotides; D006023:Glycoproteins; D009838:Oligodeoxyribonucleotides; D009841:Oligonucleotides; D008623:Mercaptoethanol; D004247:DNA; D008628:Mercury