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Nucleic Acids Research, 1982, Vol. 10, No. 13 4009-4025
© 1982

MOLECULAR BIOLOGY

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

James L. Hartley*, Kenneth K. Chen and John E. Donelson

Department of Biochemistry and Diabetes and Endocrinology Research Core Center, University of Iowa Iowa City, IA 52242, USA

Received February 8, 1982. Revised May 17, 1982. Accepted May 25, 1982.

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 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.

*Present address: Bethesda Research Labs., Inc., P.O. Box 6009, Gaithersburg, MD 20877, USA


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