DNA sequencing with a T7-type gene 6 exonuclease

Details DNA sequencing with a T7-type gene 6 exonuclease DNA sequencing with a T7-type gene 6 exonuclease

1992-08-06

1996-05-14

Jones, W. Gary

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving nucleic acid

435 912, 435 915, 436 94, 536 2433, 935 17, 935 77, 935 78, C12Q 168, C12P 1934, C07H 2104

Patent

active

055166334

ABSTRACT:
Method for determining the nucleotide base sequence of DNA present in a lambda virus coat which is not replicable in a bacterial cell independent of any lambda DNA within that coat. The method includes preparing a lambda phage preparation containing the DNA, purifying the nucleic acid from the lambda phage to provide purified nucleic acid, and contacting that purified nucleic acid with a T7-type gene 6 exonuclease to allow the exonuclease to remove at least a portion of one strand of the DNA. The method further includes providing a primer able to hybridize with the other strand of the DNA complementary to the portion of the one strand and contacting the primer with the other strand of DNA in the presence of at least one deoxynucleoside triphosphate (dNTP) and at least one chain terminating agent (e.g., a dideoxynucleoside triphosphate, ddNTP) and a DNA polymerase, to allow the primer to be extended by the polymerase until extension is stopped by incorporation of the chain terminating agent.

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