Single nucleotide polymorphism analysis of highly polymorphic target sequences

1. A method for continuous monitoring of polynucleotide amplification of a target nucleic acid sequence having at least two single nucleotide polymorphisms wherein a first single nucleotide polymorphism is to be distinguished and a second single nucleotide polymorphism is not distinguished, each of said polymorphisms being in a probe region of said target nucleic acid, the method comprising:

(a) combining a sample containing said target nucleic acid with one or more oligonucleotide primers adjacent to or overlapping with said probe region of the target sequence, a polymerizing enzyme, nucleotide substrates, and an oligonucleotide conjugate having a formula:

 wherein M is a minor groove binder; the subscript t is 0 or 1; Q is a quencher; W is a linking group; K is a bond or a linking group; Fl is a fluorophore; and [A-B] n represents a nucleic acid oligomer having n units, wherein n is an integer of from 5 to 50; each A independently represents a nucleic acid backbone component selected from the group consisting of a sugar phosphate backbone, a modified sugar phosphate backbone, a locked nucleic acid backbone, and a peptidic backbone; and each B independently represents a nucleic acid base, a modified base or a base analog and wherein the base at the site complementary to said second single nucleotide polymorphism, is a universal or promiscuous (indiscriminative) base and the oligonucleotide portion has a sequence complementary to a portion of the target sequence being amplified, to provide a mixture;

(b) incubating the mixture under conditions favorable for polymerization; and

(c) continuously monitoring the amplification by monitoring the fluorescence produced upon conjugate hybridization to the amplified target.

2. The method of claim 1 , wherein the minor groove binder (M) is selected from the group consisting of CC1065 analogs, lexitropsins, distamycin, netropsin, berenil, duocarmycin, pentamidine, 4,6-diamino-2-phenylindole and pyrrolo[2,1-c][1,4]benzodiazepine analogs.

3. The method of claim 1 , wherein the minor groove binder (M) is a substituted dihydrocyclopyrroloindole triamide (DPI 3 ).

4. The method of claim 1 , wherein the minor groove binder (M) is attacked to the 5′ end of the oligonucleotide conjugate.

5. The method of claim 1 , wherein the minor groove binder (M) is attached to the 3′ end of the oligonucleotide conjugate.

6. The method of claim 1 , wherein Q is a quencher with an absorption spectra between about 400 to 800 nm, and Fl is a fluorophore with emission wavelengths between about 400 to 800 nm.

7. The method of claim 1 , wherein said fluorophore (Fl) is selected from the group consisting of coumarins, resorufins, xanthenes, benzoxanthenes, cyanines and bodipy analogs.

8. The method of claim 1 , wherein said quencher (Q) is selected from the group consisting of mono azo and bis azo dyes.

9. The method of claim 1 , wherein said sugar phosphate backbone (A) comprises 8-25 nucleotides in length.

10. The method of claim 1 , wherein the fluorescence is produced directly upon hybridization of the probe with said target nucleic acid sequence.

11. The method of claim 1 , wherein the fluorescence is produced upon enzymatic cleavage of the quencher or the fluorophore from the conjugate probe upon hybridization with a target nucleic acid sequence.

12. The method of claim 1 , wherein the universal base is selected from the group provided in Table 1.

13. The method of claim 1 , wherein the promiscuous base is selected from the group provided in Table 2.

14. The method of claim 1 , wherein the promiscuous (indiscriminative) base has the formula:

wherein R y and R z are independently selected from the group consisting of OH and NH 2 and at least one of R y or R z is NH 2 ; and R x is a member selected from the group consisting of H, (C 1 -C 12 )alkyl, (C 2 -C 12 )alkenyl, (C 2 -C 12 )alkynyl, (C 2 -C 12 )heteroalkyl, (C 3 -C 12 )heteroalkenyl, (C 3 -C 12 )heteroalkynyl, —O—(C 1 -C 12 )alkyl, —O—(C 2 -C 12 )alkenyl, —O—(C 2 -C 12 )alkynyl, —S—(C 1 -C 12 )alkyl, —S—(C 2 -C 12 )alkenyl, —S—(C 2 -C 12 )alkynyl, heterocyclyl(C 1 -C 12 )alkyl, heterocyclyl(C 2 -C 12 )alkenyl, heterocyclyl(C 2 -C 12 )alkynyl, aryl(C 1 -C 12 )alkyl, aryl(C 2 -C 12 )alkenyl, aryl(C 2 -C 12 )alkynyl, aryl, heterocyclyl, halogen, —CN and —CONH 2 , wherein the aliphatic portions of R x are further substituted with one or two hydroxy, amino or halogen groups; and the wavy line indicates the point of attachment to the remainder of the probe.