Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Patent
1990-06-26
1991-07-02
Wax, Robert A.
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
435 91, 436501, 436 94, G01N 33566, G01N 3300, C12Q 168
Patent
active
050285254
ABSTRACT:
A method is provided for producing single stranded non-self-complementary nucleic acid probes, and for treating target DNA for use therewith. Probe is constructed by treating DNA with a restriction enzyme and an exonuclease to form template/primers for a DNA polymerase. The digested strand is resynthesized in the presence of labeled nucleoside triphosphate precursor. Labeled single stranded fragments are separated from the resynthesized fragments to form the probe. Target DNA is treated with the same restriction enzyme used to construct the probe, and is treated with an exonuclease before application of the probe. The method significantly increases the efficiency and specificity of hybridization mixtures by increasing effective probe concentration by eliminating self-hybridization between both probe and target DNAs, and by reducing the amount of target DNA available for mismatched hybridizations.
REFERENCES:
Schmidt, "Exonuclease Digestion of Chromosomces for in situ Hybridization", Nucleic Acids Research, vol. 16 (#21), 1988, p. 10381.
Workman et al., "Necleoprotein Hybridization: A Method . . . ", Biochemistry, 1985, 24, 7486-7497.
Old and Primrose, Studies in Microbiology, vol. 2, Principles of Gene Manipulation, ed. Carr et al. (University of California Press, Berkeley and Los Angeles, 1981), Chapter 6 Cloning Strategies, pp. 89-90.
James and Leffak, "Replacement Synthesis Labeling of DNA Molecules in vitro Using the Escherichia Coli Exonuclease III/DNA Polymerase I Enzyme Pair", Anal. Biochem; 141: 33-37 (1984).
Liao et al., "Cloning of Rat .alpha.-Fetoprotein 3'-Terminal Complementary Deoxyribonucleic Acid Sequences and Preparation of Radioactively Labeled Hybridization Probes from Clone Deoxyribonucleic Acid Inserts", Biochem; 20: 1646-1652 (1981).
Langer et al., "Enzymatic Synthesis of Biotin-Labeled Polynucleotides: Novel Nucleic Acid Affinity Probes", PNAS (U.S.A.), 78 (11): 6633-6637.
Gray Joe W.
Pinkel Daniel
Lauder Leona L.
Macevicz Stephen C.
Regents of the University of California
Sartorio Henry P.
Steffe Eric
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