US 5,688,669 A · Methods for nucleic acid detection, sequencing, and cloning using exonuclease

Abstract

The present invention provides a method of detecting the presence of a nucleotide sequence within a double-stranded DNA in a sample comprising: a. digesting the double-stranded DNA with an exonuclease which converts at least a portion of the double-stranded DNA to single-stranded DNA, b. binding the single-stranded DNA with a nucleic acid probe which selectively hybridizes with the single-stranded DNA, and c. detecting hybridization between the single-stranded DNA and the nucleic acid probe, the existence of hybridization indicating the presence of the nucleotide sequence within the double-stranded DNA in the sample. The present invention further provides a method of detecting the presence of a nucleotide sequence in a sample comprising DNA which is the product of a DNA amplification technique. The invention also provides methods of sequencing and cloning using exonuclease.

Claims10

Dependent claims

A method of adapting a nucleic acid for sequencing comprising:

a. amplifying the nucleic acid using a first primer containing deoxyuridine monophosphate and a second primer not containing deoxyuridine monophosphate to yield a double-stranded DNA with a polyuridylated 5' end on one strand;
b. Partially disassociating the resultant double-stranded DNA with uracil DNA glycosylase to form a 3' overhang on the polyuridylated 5' end;
c. digesting the double-stranded DNA with a 3' exonuclease which digests only the 3' strand from the end opposite the polyuridylated 5' end for a time sufficient to convert at least a portion of the double-stranded DNA to a single-stranded DNA;
and
d. extending the partially digested 3' strand with a polymerase such that sequencing by a dideoxynucleotide process can be performed on the single-stranded DNA.

2 more elements

Dependencies are inferred from the claim text.

Methods for nucleic acid detection, sequencing, and cloning using exonuclease

Life of this patent

Methods for nucleic acid detection, sequencing, and cloning using exonuclease

Life of this patent

Abstract

Claims10