DNA sequencing with Thermus aquaticus DNA polymerase and direct sequencing of polymerase chain reaction-amplified DNA.
The highly thermostable DNA polymerase from Thermus aquaticus (Taq) is ideal for both manual and automated DNA sequencing because it is fast, highly processive, has little or no 3'-exonuclease activity, and is active over a broad range of temperatures. Sequencing protocols are presented that produce readable extension products greater than 1000 bases having uniform band intensities. A combination of high reaction temperatures and the base analog 7-deaza-2'-deoxyguanosine was used to sequence through G + C-rich DNA and to resolve gel compressions. We modified the polymerase chain reaction (PCR) conditions for direct DNA sequencing of asymmetric PCR products without intermediate purification by using Taq DNA polymerase. The coupling of template preparation by asymmetric PCR and direct sequencing should facilitate automation for large-scale sequencing projects.
Innis
M A
MA
Department of Microbial Genetics, Cetus Corporation, Emeryville, CA 94608.
Myambo
K B
KB
Gelfand
D H
DH
Brow
M A
MA
D001322:Autoanalysis; D001483:Base Sequence; D004247:DNA / Q000032:analysis*; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism*; D003849:Deoxyguanosine / Q000031:analogs & derivatives; D004591:Electrophoresis, Polyacrylamide Gel; D009710:Nucleotide Mapping / Q000379:methods*; D013696:Temperature; D013698:Templates, Genetic; D013824:Thermus / Q000201:enzymology
C045550:7-deaza-2'-deoxyguanosine; D004247:DNA; D004259:DNA-Directed DNA Polymerase; D003849:Deoxyguanosine