Nucleic acid detection by repetitive synthesis and degradation o

Details Nucleic acid detection by repetitive synthesis and degradation o Nucleic acid detection by repetitive synthesis and degradation o

1996-01-29

1998-12-15

Zitomer, Stephanie W.

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving nucleic acid

435 911, 435 912, 435 9152, 435193, 435194, 435196, 536 243, C12Q 168, C12P 1934, C07H 2102

Patent

active

058494871

ABSTRACT:
The present invention relates to a method of detecting a polynucleotide, comprising hybridizing a polynucleotide of known nucleotide sequence with a nuclease-resistant oligonucleotide primer having a sequence complementary to a part of the polynucleotide, then adding at least one kind of deoxynucleoside triphosphate, DNA polymerase and nuclease thereto, synthesizing a complementary strand being a nucleotide species located adjacent to the 3'-terminal of the primer and complementary to the polynucleotide, followed by decomposition thereof, the synthesis and decomposition of the complementary strand being repeated one or more times, and detecting the resulting pyrophosphoric acid or deoxynucleoside monophosphate. The present invention also includes a detection kit used for this method of detecting a polynucleotide.

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Hershfield et al, (1972), "Hydrolysis of template and newly synthesized deoxyribonucleic acid by the 3' to 5' exonuclease activity of the T4 Deoxyribonucleic acid polymerase" J. Biol. Chem. 247(11):3393-3404.
Tang et al, (Jun. 1993), "Self stabilized antisense oligodexynucleotide phosphorothioate properties and anti-HIV activity", Nucleic Acids Res. 21(11):2729-2735.
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Ott et al, (1987), "Protection of oligonucleotide primers against degradation by DNA polymerase I", Biochemistry 26:8237-8241.
Gupta et al, (1984), "The effect of the 3' 5' thiophosphoryl linkage on the exonuclease activities of T4 polymerase and the Klenow fragment", Nucleic Acids Res. 12(14):5897-5911.
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