Strategy and methods for directly sequencing cosmid clones.
The primer-directed enzymatic sequencing method for sequencing double-stranded DNA templates has made possible the development of new strategies for directly sequencing large DNA molecules. Toward this goal, we have developed a strategy and the necessary techniques to obtain the complete sequence of cosmid clones (double-stranded DNA molecules in the size range of 50 kb). Our present strategy uses the chemical sequencing method to obtain sequence initiation points internal to a cosmid insert and the primer-directed enzymatic DNA sequencing method to extend these sequence contigs. As part of this development we added a nucleotide "chase" solution to the standard T7 sequencing protocol and included the use of both [alpha-32P]-dATP and -dCTP for labeling. With these modifications our double-stranded cosmid DNA sequencing reactions routinely extend well beyond 1000 bp, and film exposure times are kept to a minimum (24 to 48 h). We can routinely separate sequenced DNA fragments, using a 1-m gel system, which can be accurately read (with less than 0.5% error) to distances of 800 bp or more, from the oligomer primer. The strategy and procedures presented here allow the complete sequence of a cosmid clone to be obtained without subcloning.
Siemieniak
D R
DR
Molecular Biology Research-Unit 7242, Upjohn Company, Kalamazoo, Michigan 49007.
Sieu
L C
LC
Slightom
J L
JL
D001483:Base Sequence; D003001:Cloning, Molecular; D003360:Cosmids; D004247:DNA; D004259:DNA-Directed DNA Polymerase; D005821:Genetic Techniques; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction
D004247:DNA; C034970:bacteriophage T7 induced DNA polymerase; D004259:DNA-Directed DNA Polymerase