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 steps of:
obtaining a sample comprising a nucleic acid, the nucleic acid comprising a target nucleic acid;
amplifying the target nucleic acid using a pair of primers complementary to the target nucleic acid, the pair comprising a first primer comprising the nucleotide sequence of SEQ ID NO: 1 and wherein nucleotides 1-4 are modified nucleotides that are not susceptible to hydrolysis by a 5′ to 3′ double stranded nucleic acid specific exonuclease and a second primer comprising SEQ ID NO: 2, wherein the amplification provides double stranded amplicons products comprising a first strand comprising the modified primer and a downstream amplified region and a second strand;
incubating the double stranded amplicons with a 5′ to 3′ double stranded nucleic acid specific exonuclease which hydrolyses the second strand but does not hydrolyse the amplified region of the first strand, to provide single stranded nucleic acid products comprising the amplified region of the first strand;
exposing the single stranded nucleic acid products to a plurality of electrochemically labelled probes designed to specifically hybridize to the single stranded nucleic acid products, thereby producing labelled hybridized nucleic acid products;
exposing the labelled hybridized nucleic acid products to the exonuclease which hydrolyses the hybridized labelled probes causing a detectable electrochemical signal change from each of the hybridized labelled probes; and
allowing a sufficient period of time for one after another one of the plurality of electrochemically labelled probes to hybridize with a same one of the single stranded nucleic acid products, thereby generating at least three detectable electrochemical signal changes from each of the single stranded nucleic acid products; and
detecting each of the single stranded nucleic acid products multiple times.
2. The method of claim 1 further comprising steps of:
incubating the sample comprising nucleic acid with uracil-N-glycosylase to hydrolyze nucleic acid containing uracil, thereby producing a sample free of contaminating nucleic acid containing uracil; and
performing the nucleic acid amplification step with dUTP in the absence of dTTP.
3. The method of claim 1 , wherein the nucleic acid amplification is achieved using PCR.
4. The method of claim 1 , wherein the sample is a human sample, or a cellular sample.
5. The method of claim 1 , wherein the at least one modified nucleotide comprises at least one modified sugar moiety, at least one modified intenucleoside linkage and/or at least one modified nucleobase.
6. The method of claim 5 , wherein the at least one modified sugar moiety is a 2′-O-methyl sugar moiety; and/or the at least one modified intenucleoside linkage is a phosphorothioate linkage.
7. The method of claim 1 , wherein the modified primer comprises at at least 4, at least 5, at least 6, at least 7, at least 8, at least 9 or at least 10 modified nucleotides, wherein optionally the modified primer comprises 3 or 4 phosphorothioate linkages.
8. The method of claim 1 , wherein the primers specifically hybridise to a Chlamydia trachomatis nucleic acid.
9. The method of claim 1 , wherein the exonuclease is T7 exonuclease.
10. The method of claim 1 , wherein the electrochemical label of the electrochemically labelled probes is a ferrocene label.
11. A method of detecting a nucleic acid of interest in a sample comprising nucleic acids, the method comprising:
a) amplifying the nucleic acid of interest in the sample using a modified first primer comprising the nucleotide sequence of SEQ ID NO: 1 and wherein nucleotides 1-4 are modified nucleotides and a second primer comprising SEQ ID NO: 2, producing a double stranded nucleic acid having a first strand comprising the modified primer and the amplified nucleic acid of interest and a second strand;
b) denaturing the double stranded nucleic acid with a 5′ to 3′ double stranded nucleic acid specific exonuclease, producing a single stranded nucleic acid product comprising the amplified nucleic acid of interest;
c) hybridizing the single stranded nucleic acid product to more than one of a plurality of labelled probes complementary to the single stranded nucleic acid, producing a labelled nucleic acid product;
d) exposing the labelled nucleic acid product to a 5′ to 3′ double stranded nucleic acid specific exonuclease to hydrolyze the labelled probe, thereby producing a signal and allowing a sufficient period of time for at least another two of the plurality of labelled probes to hybridize to the single stranded nucleic acid; and
e) detecting multiple signals from the amplified nucleic acid of interest.