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

A method of detecting the presence of a point mutation 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. hybridizing the single-stranded DNA with i) a first nucleic acid probe containing a nucleotide complementary to the nucleotide of the point mutation at a first end and adapted with a moiety which can be captured to a solid support, and ii) a second nucleic acid probe labeled with a detectable moiety and which can hybridize at a second end with the single-stranded DNA adjacent the hybridized first end of the first nucleic acid probe;
• ligating the hybridized first and second nucleic acid probes;
• d. capturing the moiety on the first nucleic acid probe to the solid support;
• e. denaturing the ligated first and second nucleic acid probes from the hybridized single-stranded DNA;
• f. removing uncaptured labeled probe;
• and
• g. detecting captured detectable moiety, the presence of captured detectable moiety indicating the presence of the point mutation within the double-stranded DNA in the sample.

A method of detecting the presence of a point mutation within a double-stranded DNA at a level of one copy of the point mutation per at least about 75,000 normal copies 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. hybridizing the single-stranded DNA with i) a first nucleic acid probe containing a nucleotide complementary to the nucleotide of the point mutation at a first end and adapted with a moiety which can be captured to a solid support, and ii) a second nucleic acid probe labeled with a detectable moiety and which can hybridize at a second end with the single-stranded DNA adjacent the hybridized first end of the first nucleic acid probe;
• c. ligating the hybridized first and second nucleic acid probes;
• capturing the moiety on the first nucleic acid probe to the solid support;
• e. denaturing the ligated first and second nucleic acid probes from the hybridized single-stranded DNA;
• f. removing uncaptured labeled probe;
• and
• g. detecting captured detectable moiety, the presence of captured detectable moiety indicating the presence of the point mutation per at least about 75,000 normal copies in the sample.

A kit comprising:

• a) an exonuclease;
• b) a first nucleic acid probe which binds to target DNA and which is adapted with a capture moiety;
• c) a second nucleic acid probe which binds to target DNA adjacent the first probe and which is labeled with a detectable moiety;
• and
• d) a ligase.

A method of detecting the presence of a point mutation 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. hybridizing the single-stranded DNA with i) a first nucleic acid probe containing a nucleotide at a first end complementary to the nucleotide of the wild type nucleic acid sequence at a nucleotide position suspected to be a point mutation and adapted with a moiety which can be captured to a solid support, and ii) a second nucleic acid probe labeled with a detectable moiety and which can hybridize at a second end with the single-stranded DNA adjacent the hybridized first end of the first nucleic acid probe;
• c. ligating the hybridized first and second nucleic acid probes;
• d. capturing the moiety on the first nucleic acid probe to the solid support;
• e. denaturing the ligated first and second nucleic acid probes from the hybridized single-stranded DNA;
• f. removing uncaptured labeled probe;
• and
• g. detecting captured detectable moiety, the absence of captured detectable moiety indicating the presence of the point mutation within the double-stranded DNA in the sample.