Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Patent
1989-10-02
1994-10-11
Fleisher, Mindy B.
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
536 243, 536 2433, 935 77, 935 78, C12Q 168, C07H 2104, C12N 1500
Patent
active
053546563
ABSTRACT:
An improved method of determining the nucleotide base sequence of DNA is disclosed. The method comprises preparing a DNA substrate comprising a set of molecules, each having a template strand and a primer strand, wherein the 3' ends of the primer strands terminate at about the same nucleotide position on the template strands of the molecules within each set. DNA synthesis is induced to obtain labeled reaction products comprising newly synthesized DNA complementary to the template strands using the 3' ends of the primer strands to prime DNA synthesis, labeled nucleoside triphosphates, and at least one modified nucleoside triphosphate, wherein the modified nucleoside triphosphate is selected to substantially protect newly synthesized DNA from cleavage. The labeled reaction products are cleaved at one or more sites to obtain labeled DNA fragments wherein the newly synthesized DNA is substantially protected from cleavage at the selected site or sites. The labeled DNA fragments are separated and their nucleotide base sequence is identified by suitable means.
REFERENCES:
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Maniatis et al, Molecular Cloning: A Laboratory Manual, 1982, Cold Spring Harbor Laboratory, p. 102.
Sanger et al, Proc. Natl. Acad. Sci. USA, Dec. 1977, vo. 74, No. 12, pp. 5463-5467.
Leader et al., DNA, 5:235-238 (1986).
Church et al., Proc. Natl. Acad. Sci., 81:1991-1995 (1984).
Guo et al., Nucleic Acids Res., 10:2065-2085 (1982).
Guo et al., Meth. Enz., 100:60-97.
Maxam and Gilbert, Meth. Enzymol., 65:499-561 (1977).
Huse William D.
Sorge Joseph A.
Fleisher Mindy B.
Sherman Ronni L.
Stratagene
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