Method for detecting a nucleic acid sequence

1 . A method for detecting one or more target sequences in a sample by amplification, the method comprising:

a) providing an aqueous composition comprising:

i) one or more forward oligonucleotide primers having a 5′ region which is a tag sequence and a 3′ region specific for a target sequence;

ii) a reverse oligonucleotide primer, such that a forward oligonucleotide primer from the one or more forward oligonucleotide primers and the reverse primer operably form a primer pair;

iii) one or more probes having a reporter label and a quencher label, each of the one or more probes comprising a sequence having at least 90% identity to the tag sequence of the forward oligonucleotide primer from the one or more forward oligonucleotide primers; and

iv) one or more enhancer oligonucleotide primers comprising a sequence having at least 90% identity to the sequence of a probe from the one or more probes;

b) incubating the primers and the probes with the sample and at least a DNA polymerase, wherein the DNA polymerase is a 5′ nuclease-deleted DNA polymerase; and

c) performing polymerase chain reaction (PCR) in a single reaction master mix comprising the sample to generate tagged nucleic acids, whereby the probes can bind to the tagged nucleic acids and label the one or more target sequences by generating a signal; wherein the reporter label is detectable spectroscopically, photochemically, biochemically, immunochemically, or chemically and is selected from the group consisting of an enzyme, enzyme substrate, radioactive atom, fluorescent dye, chromophore, chemiluminescent label, and a ligand having a specific binding partner or partners.

2 . The method according to claim 1 , wherein the one or more enhancer oligonucleotide primers comprise at least two distinct regions, each of the two distinct regions comprising a sequence having at least 90% identity to a sequence of the one or more probes.

3 . The method according to claim 2 , wherein the two distinct regions are separated by a linker region.

4 . The method according to claim 3 , wherein the linker region is 1 to 10 or 2 to 5 oligonucleotides in length.

5 . The method according to claim 1 , wherein the sequence having at least 90% identity to the tag sequence of the forward oligonucleotide primer from the one or more oligonucleotide primers is a sequence having at least 95%, 98% or 99% identity to the tag sequence of the forward oligonucleotide primer from the one or more forward oligonucleotide primers or a sequence which is 100% identical to the tag sequence of the forward oligonucleotide primer from the one or more forward oligonucleotide primers.

6 . The method according to claim 1 , wherein the sequence having at least 90% identity to the tag sequence of the probe from the one or more probes is a sequence having at least 95%, 98% or 99% identity to the tag sequence of the probe from the one or more probes or a sequence which is 100% identical to the sequence of the probe from the one or more probes.

7 . The method according to claim 1 , wherein at least 4, 20, or 30 cycles of PCR are performed in the single reaction master mix.

8 . The method according to claim 1 , further comprising: d) measuring the signal generated by the binding of the probes to the tagged nucleic acids.

9 . The method according to claim 8 , wherein the measurement is made in real time or at the end of the reaction.

10 . The method according to claim 1 , wherein the composition further comprises a quaternary ammonium salt.

11 . The method according to claim 10 , wherein the quaternary ammonium salt is a tetramethylammonium salt, optionally wherein the tetramethylammonium salt is tetramethylammonium chloride (TMAC).

12 . The method according to claim 1 , wherein each of the one or more probes comprises a portion at or adjacent to its 3′ terminus which is complementary to a portion at or adjacent to its 5′ terminus, optionally wherein:

(a) the its 3′ terminal portion and/or the its 5′ terminal portion have a length of at least 4, at least 5, at least 6 or at least 7 nucleotides, and/or

(b) wherein the its 3′ terminal portion and the its 5′ terminal portion have at least 90% or 100% complementarity.

13 . The method according to claim 1 , wherein each of the one or more enhancer oligonucleotide primers comprises a portion at or adjacent to its 3′ terminus which is complementary to a portion at or adjacent to its 5′ terminus, optionally wherein

(a) the its 3′ terminal portion and/or the its 5′ terminal portion have a length of at least 4, at least 5, at least 6 or at least 7 nucleotides, and/or

(b) wherein the its 3′ terminal portion and the 5′ terminal portion: (i) have at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% or 100% complementarity, or (ii) comprise one, two or three mismatches.

14 . The method according to claim 1 , wherein:

(a) the 3′ terminus or the 5′ terminus of the one or more probes further comprises an overhang portion of one, two or three nucleotides which does not hybridize to nucleotides on the 5′ or 3′ terminal portion of the one or more probes respectively; or

(b) the one or more enhancer oligonucleotide primers have a hairpin structure, the one or more probes have a hairpin structure, and the hairpin structure in the one or more enhancer oligonucleotide primers is less stable than the hairpin structure in the one or more probes.

15 . The method according to claim 1 , wherein the reporter label is a fluorescent dye and is detectable spectroscopically or photochemically.

16 . The method according to claim 1 , wherein the reporter label is an enzyme and is detectable photochemically, spectroscopically, biochemically, immunochemically, or chemically.

17 . The method according to claim 1 , wherein the reporter label is an enzyme substrate and is detectable photochemically, spectroscopically, biochemically, immunochemically, or chemically.

18 . The method according to claim 1 , wherein the reporter label is a radioactive atom and is detectable photochemically or chemically.

19 . The method according to claim 1 , wherein the reporter label is a chromophore and is detectable spectroscopically or photochemically.

20 . The method according to claim 1 , wherein the reporter label is a chemiluminescent label and is detectable spectroscopically or photochemically.

21 . The method according to claim 1 , wherein the reporter label is a ligand having a specific binding partner or partners and is detectable biochemically, chemically, or immunochemically.

22 . A method for detecting one or more target sequences in a sample by amplification, the method comprising:

a) providing an aqueous composition comprising:

i) one or more forward oligonucleotide primers having a 5′ region which is a tag sequence and a 3′ region specific for a target sequence;

ii) a reverse oligonucleotide primer, such that a forward oligonucleotide primer from the one or more forward oligonucleotide primers and the reverse primer operably form a primer pair;

iii) one or more probes having a reporter label and a quencher label, each of the one or more probes comprising a sequence having at least 90% identity to the tag sequence of the forward oligonucleotide primer from the one or more forward oligonucleotide primers; and

iv) one or more enhancer oligonucleotide primers comprising a sequence having at least 90% identity to the sequence of a probe from the one or more probes;

b) incubating the primers and the probes with the sample and at least a DNA polymerase, wherein the DNA polymerase is a 5′ nuclease-deleted DNA polymerase; and

c) performing polymerase chain reaction (PCR) in a single reaction master mix comprising the sample to generate tagged nucleic acids, whereby the probes can bind to the tagged nucleic acids and label the one or more target sequences by generating a signal;

wherein the reporter label is a fluorescent dye and is detectable spectroscopically or photochemically;

wherein the reporter label is an enzyme and is detectable photochemically, spectroscopically, biochemically, immunochemically, or chemically;

wherein the reporter label is an enzyme substrate and is detectable photochemically, spectroscopically, biochemically, immunochemically, or chemically;

wherein the reporter label is a radioactive atom and is detectable photochemically or chemically;

wherein the reporter label is a chromophore and is detectable spectroscopically or photochemically;

wherein the reporter label is a chemiluminescent label and is detectable spectroscopically or photochemically; or

wherein the reporter label is a ligand having a specific binding partner or partners and is detectable biochemically, chemically, or immunochemically.

23 . The method according to claim 1 , further comprising increasing the signal-to-noise ratio of the signal generated in step c) by adding a quaternary ammonium salt.

24 . The method according to claim 23 , wherein:

(a) the quaternary ammonium salt is a tetramethylammonium salt, optionally wherein the tetramethylammonium salt is tetramethylammonium chloride (TMAC); and

(b) the presence of TMAC increases the signal-to-noise ratio of the signal from the probes.