Nucleic acid enrichment and detection

A method for altering the ratio of a first nucleic acid molecule to a second nucleic acid molecule in a sample, wherein the sample comprises at least the first and second nucleic acid molecules, the method comprising the steps of:

• a. introducing the sample comprising one or more nucleic acid analytes to a first reaction mixture comprising:
• i. a single-stranded probe oligonucleotide A0 wherein the probe has greater complementarity to a first nucleic acid molecule relative to a second nucleic acid molecule;
• ii. a pyrophosphorolysing enzyme;
• and
• iii. a source of pyrophosphate ion;
• wherein A0 anneals better to the first nucleic acid molecule relative to the second nucleic acid molecule to create an at least partially double-stranded intermediate product in which the 3′ end of A0 forms a double-stranded complex with said first nucleic acid molecule and A0 is pyrophosphorolysed in the 3′-5′ direction from its 3′ end, whilst any A0 that has annealed to the second nucleic acid molecule is pyrophosphorolysed in the 3′-5′ direction to a lesser extent relative A0 that annealed to first nucleic acid molecule;
• b. selectively denaturing any shortened A0 sequence complexes which were annealed to the first nucleic acid molecule;
• and
• c. separating A0, and thereby any second nucleic acid sequences remaining annealed thereto, from the first nucleic acid sequences not annealed to A0, thereby altering the ratio of the first nucleic acid molecule to the second nucleic acid molecule.

A method for increasing the ratio of a first nucleic acid sequence to a second nucleic acid sequence in a sample, wherein the sample comprises the first and second nucleic acid molecules, the method comprising the steps of:

• a. introducing the sample comprising one or more nucleic acid analytes to a first reaction mixture comprising:
• i. a single-stranded probe oligonucleotide A0 wherein said probe has greater complementarity to one of the first or second nucleic acid molecules relative to the other of the first or second nucleic acid molecules;
• ii. a pyrophosphorolysing enzyme;
• and
• iii. a source of pyrophosphate ion;
• wherein A0 anneals better to one of the nucleic acid molecules relative to the other of the nucleic acid molecules to create an at least partially double-stranded intermediate product in which A0 forms a double-stranded complex with said nucleic acid molecule and A0 is pyrophosphorolysed in the 3′-5′ direction from its 3′-end, whilst any A0 that has annealed to a lesser degree to the other nucleic acid relative to said nucleic acid molecule is pyrophosphorolysed in the 3′-5′ direction to a lesser extent relative to A0 that is hybridized to said nucleic acid molecule;
• b. separating A0 sequence complexes formed with said nucleic acid molecule which were annealed by:
• i. allowing the strands of said complex to separate as a consequence of the pyrophosphorolysis reaction;
• or
• ii. heating the reaction mixture to a temperature sufficient for the stands of said complex to separate but which is below the temperature required for the stands of any A0 which annealed to a lesser degree to the other nucleic acid sequence to separate;
• and
• c. separating A0, and thereby any nucleic acid sequences remaining annealed thereto, from any nucleic acid sequences not annealed to A0.