Monochrome multiplex quantitative PCR

1. A method for determining in a target sample the copy number of a first target nucleic acid and a second target nucleic acid, the method comprising: a) contacting a first target nucleic acid with a first primer set and a second target nucleic acid with a second primer set and adding a single detection label to form a reaction mixture in a homogenous system, wherein one of the primers of the first primer set is blocked from priming the first target nucleic acid and wherein one of the primers of the first primer set is blocked from priming the first target nucleic acid;

wherein the primer blocked from priming the first target nucleic acid comprises a mismatched base at its 3′ end″ and wherein the first target nucleic acid comprises a tandem repent sequence; b) amplifying by polymerase chain reaction the first target nucleic acid with the first primer set to form a first amplicon having a first melting temperature (Tm) and amplifying by polymerase chain reaction the second target nucleic acid with the second primer set to form a second amplicon having a second Tm, wherein said second Tm is higher than said first Tm; c) during the polymerase chain reaction, determining the amount of the detection label at a first acquisition temperature, wherein said first signal acquisition temperature is below said first Tm; d) increasing the temperature of the reaction mixture to a second signal acquisition temperature and determining the amount of the detection label, wherein said second signal acquisition temperature is above said first TM and below said second Tm; e) repeating steps (b) through (d) at least one time; and f) determining the copy number of said first and said second target nucleic acids.

2. The method of claim 1 , wherein the copy number of the first target nucleic acid sequence is greater than the copy number of the second target nucleic acid sequence.

3. The method of claim 1 , wherein the amount of the detection label is detected at said first and said second signal acquisition temperatures during each of said amplification steps.

4. The method of claim 1 wherein the difference between the first Tm and the second Tm is at least 4 degrees Celsius.

5. The method of claim 1 , wherein at least one of the primers in said first primer set comprises a 5′ sequence that includes A and T nucleotides.

6. The method of claim 1 , wherein the 3′ ends of the primers of the first primer set are complementary to each other.

7. The method of claim 6 , wherein one primer of the first primer set is a mismatch primer comprising at least one mismatched nucleotide adjacent to the 3′ end of the primer, wherein said nucleotide is not complementary to the target nucleic acid, but is complementary to the 3′ terminal nucleotide of the other primer in the first primer set.

8. The method of claim 7 , wherein the extension product of the mismatch primer of the first primer set is capable of hybridizing to the other primer in the first primer set.

9. The method of claim 1 , wherein the primer blocked from priming the first target nucleic acid comprises a mismatched base at its 3′ end.

10. The method of claim 1 wherein the detection label is an intercalating dye.

11. The method of claim 1 , wherein the copy number of a first and a second target nucleic acids measures the relative amount of the first nucleic acid as compared to the second nucleic acid.

12. The method of claim 1 , wherein the first target nucleic acid is obtained from a sample.

13. The method of claim 1 , wherein the copy number of the first target nucleic acid determines the number of tandem repeat sequences present in the sample.

14. The method of claim 1 , wherein the copy number of the first target nucleic acid sequence is similar to the copy number of the second target nucleic acid sequence.

15. The method of claim 14 , wherein the polymerase chain reaction comprises at least three consecutive stages of cycles, wherein the first stage of cycles of the polymerase chain reaction comprises a polymerase chain reaction wherein the annealing temperature of the polymerase chain reaction is higher than the annealing temperature of the second stage of cycles, wherein the second stage of cycles of the polymerase chain reaction comprises a polymerase chain reaction wherein the annealing temperature of the polymerase chain reaction is lower than the annealing temperature of the first stage of cycles, and wherein the third stage of cycles of the polymerase chain reaction comprises a polymerase chain reaction wherein the annealing temperature of the polymerase chain reaction is lower than the annealing temperature of the first stage of cycles and higher than the annealing temperature of the second stage of cycles.

16. The method of claim 15 , wherein only the first amplicon is formed during the first stage of cycles of the polymerase chain reaction.

17. The method of claim 15 , wherein only the second amplicon is formed during the second stage of cycles of the polymerase chain reaction.

18. The method of claim 15 , wherein both the first and second amplicons are formed during the third stage of cycles of the polymerase chain reaction.

19. The method of claim 15 , wherein the amplification step is repeated until the detection label is determined at said second signal acquisition temperature.

20. The method of claim 15 , wherein the amount of the detection label is detected at said first and said second signal acquisition temperatures during each of said amplification steps.

21. The method of claim 15 , wherein the difference between the first Tm and the second Tm is at least 4 degrees Celsius.

22. The method of claim 15 , wherein at least one of the primers in said first primer set comprises a 5′ sequence that includes A and T nucleotides.

23. The method of claim 15 , wherein the 3′ ends of the primers of the first primer set are complementary to each other.

24. The method of claim 23 , wherein one primer of the first primer set is a mismatch primer comprising at least one mismatched nucleotide adjacent to the 3′ end of the primer, wherein said nucleotide is not complementary to the target nucleic acid, but is complementary to the 3′ terminal nucleotide of the other primer in the first primer set.

25. The method of claim 24 , wherein the extension product of the mismatch primer of the first primer set is capable of hybridizing to the other primer in the first primer set.

26. The method of claim 15 , wherein one of the primers of the first primer set is blocked from priming the first target nucleic acid.

27. The method of claim 26 , wherein the primer blocked from priming the first target nucleic acid comprises a mismatched base at its 3′ end.

28. The method of claim 15 , wherein the detection label is an intercalating dye.

29. The method of claim 15 , wherein the copy number of a first and a second target nucleic acids measures the relative amount of the first nucleic acid as compared to the second nucleic acid.

30. The method of claim 15 , wherein the first target nucleic acid comprises a tandem repeat sequence.

31. The method of claim 15 , wherein the first target nucleic acid is obtained from a sample.

32. The method of claim 31 , wherein the copy number of the first target nucleic acid determines the number of tandem repeat sequences present in the sample.

33. The method of claim 1 , wherein the amount of detection label determined during the first and said second signal acquisition temperatures are compared to a control.

34. The method of claim 1 , wherein the amount of detection label determined during the first and said second signal acquisition temperatures are compared to a control.