Probe based nucleic acid detection
View Patent ↗The invention provides a method for detecting a target nucleotide sequence by tagging the nucleotide sequence with a nucleotide tag, providing a probe oligonucleotide with a melting temperature Tm 1 , comprising a regulatory sequence and a nucleotide tag recognition sequence; incorporating the probe oligonucleotide into the tagged polynucleotide in a polynucleotide amplification reaction, providing a regulatory oligonucleotide with a melting temperature Tm 2 , comprising a sequence segment that is at least partially complementary to the regulatory sequence, amplifying the tagged target nucleic acid sequence in a PCR amplification reaction using the probe oligonucleotide as a primer, and detecting the amplification product; wherein Tm 1 and Tm 2 are higher than the annealing temperature associated with the polynucleotide amplification reaction.
1. A method for detecting a target nucleotide sequence comprising:
a) tagging the target nucleotide sequence with a nucleotide tag sequence, thereby producing a tagged target nucleic acid sequence;
b) providing a probe oligonucleotide comprising a nucleotide tag recognition sequence complementary to the nucleotide tag sequence and a regulatory sequence 5′ to the nucleotide tag recognition sequence, wherein said probe oligonucleotide comprises a first label and has a melting temperature Tm 1 ;
c) amplifying the tagged target nucleic acid sequence in a PCR amplification reaction using the probe oligonucleotide as a primer, wherein said PCR amplification reaction is characterized by an annealing temperature Ta;
wherein the PCR amplification reaction is carried out in the presence of a regulatory oligonucleotide comprising a sequence segment that is complementary to the regulatory sequence, wherein said regulatory oligonucleotide comprises a second label and has a melting temperature Tm 2 ; and
d) detecting the product of the PCR amplification reaction;
wherein the first label and the second label constitute a fluorescent reporter/quencher pair; and wherein Tm 1 and Tm 2 are both higher than Ta.
2. The method of claim 1 wherein the nucleotide tag sequence is incorporated into the tagged target nucleic acid sequence using a PCR reaction.
3. The method of claim 1 wherein the nucleotide tag recognition sequence is exactly complementary to the nucleotide tag sequence.
4. The method of claim 1 wherein the regulatory oligonucleotide comprises a sequence segment that is exactly complementary to the regulatory sequence.
5. The method of claim 4 wherein the regulatory oligonucleotide has a length in the range of 15-45 nucleotides.
6. The method of claim 1 wherein Ta is in the range of 55-62° C.
7. The method of claim 1 wherein Ta is in the range of 60-64° C.
8. The method of claim 1 wherein Ta is in the range of 60° C-62° C.
9. The method of claim 1 wherein Tm 1 is at least 25° C. higher than the annealing temperature (Ta).
10. The method of claim 9 wherein Tm 2 is at least 2° C. higher than the annealing temperature (Ta).
11. The method of claim 10 wherein Tm 2 is in the range of 60-75° C.
12. The method of claim 8 wherein the PCR amplification reaction is carried out in a reaction volume greater than 500 nL.
13. The method of claim 12 wherein the PCR amplification reactions are carried out in a multiwell plate comprising 96-1536 wells.
14. The method of claim 8 wherein the PCR amplification reaction is carried out in a reaction volume less than 100 nL.
15. The method of claim 14 wherein the PCR amplification reaction is carried out in a microfluidic device.
16. The method of claim 14 wherein Ta is 60° C.
17. The method of claim 8 wherein the PCR amplification reaction comprises at least 20 cycles at the annealing temperature (Ta).
18. The method of claim 9 wherein Tm 2 is at least 4° C. higher than the annealing temperature (Ta).
19. The method of claim 9 wherein Tm 2 is at least 10° C. higher than the annealing temperature (Ta).
20. The method of claim 8 wherein the PCR amplification reaction is carried out in a reaction volume greater than 1 uL.