Modified primers for nucleic acid amplification and detection
A method of nucleic acid amplification involving using a first modified primer which provides protection to the amplification product from exonuclease degradation and a second primer. The method provides a double stranded nucleic acid, one strand of which is degraded by a double strand nucleic acid specific exonuclease to form a single stranded nucleic acid, which is protected from exonuclease degradation.
1. A nucleic acid amplification method comprising:
amplifying a sample, with a first primer comprising at least one modified nucleotide and a second primer to provide a double stranded nucleic acid comprising a first strand comprising the first primer and a downstream amplified region and a second strand, wherein the first primer comprises SEO ID NO: 1 or 2;
incubating the double stranded nucleic acid with a 5′ to 3′ first exonuclease that hydrolyzes the second strand but not the first strand;
hybridizing a probe comprising a label to the first strand;
hydrolyzing the probe with a second exonuclease that does not hydrolyze the first strand, to produce a detectable signal from the label; and
repeating hybridization and hydrolysis of the probe to provide an increase in the detectable signal.
2. The method of claim 1 , further comprising incubating the sample with uracil-N-glycosylase to hydrolyze nucleic acid containing uracil; and performing the amplifying step with dUTP in the absence of dTTP.
3. The method of claim 1 , wherein amplifying the sample is achieved using PCR.
4. The method of claim 1 , wherein the at least one modified nucleotide comprises at least one modified nucleobase, at least one modified sugar moiety, at least one phosphorothioate linkage, or at least one phosphoramidite linkage.
5. The method of claim 4 , wherein the at least one modified nucleotide comprises at least one modified sugar moiety.
6. The method of claim 4 , wherein the at least one modified nucleotide comprises a phosphorothioate linkage.
7. The method of claim 4 , wherein the at least one modified nucleotide comprises a phosphoramidite linkage.
8. The method of claim 1 , wherein the first primer comprises at least 4 modified nucleotides.
9. The method of claim 1 , wherein the first and/or second exonuclease is T7 exonuclease.
10. The method of claim 1 , wherein the label is an electrochemical label.
11. The method of claim 10 , wherein the electrochemical label is a ferrocene label.
12. The method of claim 1 , wherein the second primer comprises the other of SEQ ID NOS: 1 or 2.
13. A method of detecting a genetic target, the method comprising:
annealing a primer comprising SEQ ID NO: 1 or 2 to nucleic acid in a sample, the primer comprising at least one modified nucleotide;
extending the primer to create a copy of the genetic target;
hybridizing a first labeled probe to the copy; and
digesting the first labeled probe with an exonuclease to release a label from the probe and generate a detectable signal while the at least one modified nucleotide prevents the exonuclease from digesting the copy.
14. The method of claim 13 , further comprising annealing a second labeled probe to the copy and digesting the second labeled probe to increase a signal-to-noise ratio of the detectable signal.
15. The method of claim 13 , wherein the at least one modified nucleotide comprises at least one modified nucleobase, at least one modified sugar moiety, at least one phosphorothioate linkage, or at least one phosphoramidite linkage.
16. The method of claim 13 , wherein the exonuclease is T7 exonuclease.
17. The method of claim 13 , wherein the label is an electrochemical label.
18. The method of claim 17 , wherein the electrochemical label is a ferrocene label.