Dideoxy sequencing of double-stranded DNA from poly(A)-extended 3'-ends.
A simple method has been developed for sequencing double-stranded DNA by the chain termination method. The DNA to be sequenced is cut with a restriction enzyme that leaves a 3'-overhang which is extended by terminal deoxynucleotidyltransferase with limiting amounts of dATP. The sequencing reaction is then primed with an oligo(dT) primer which has a base pair "anchor" complementary to the overhang generated by the restriction enzyme. The method presented here eliminates the need for subcloning of the DNA or sequencing by chemical modification. Furthermore, sequences of more than 300 nucleotides are obtained from any 3'-overhanging restriction end.
D001345:Autoradiography; D001483:Base Sequence; D004247:DNA / Q000302:isolation & purification*; D004253:DNA Nucleotidylexotransferase; D004262:DNA Restriction Enzymes; D015252:Deoxyribonucleases, Type II Site-Specific; D009710:Nucleotide Mapping; D010957:Plasmids; D011061:Poly A / Q000032:analysis*; D013698:Templates, Genetic
D011061:Poly A; D004247:DNA; D004253:DNA Nucleotidylexotransferase; D004262:DNA Restriction Enzymes; D015252:Deoxyribonucleases, Type II Site-Specific; C029312:GGTACC-specific type II deoxyribonucleases