Photoinduced electron transfer (pet) primer for nucleic acid amplification

Abstract

This application provides photoinduced electron transfer (PET) nucleic acid molecules that can be used detect and amplify nucleic acid molecules, such as target nucleic acid molecules. These PET tags can be attached to the 5′-end of a target sequence-specific primer, thereby generating a PET primer. In particular examples, a PET tag includes a 5′-labeled nucleotide that can be quenched by at least two consecutive Gs within the tag sequence, and is unquenched when the PET tag hybridizes with its complementary nucleic acid molecule. Also disclosed are methods of using PET primers in nucleic acid amplification, such as real-time PCR.

We claim: 
     
         1 . A method of making a labeled sequence-specific primer, comprising:
 adding a photoinduced electron transfer (PET) tag to a sequence-specific primer, thereby generating a labeled sequence-specific primer,   wherein the PET tag comprises the nucleic acid sequence 5′-X 1 X 2(a) X 3 X 4(a) G x -3′ (SEQ ID NO: 1), wherein X 1  is a 5′-end labeled nucleotide, wherein X 2  and X 4  comprise a stem of a stem-loop and are nucleotide sequences of length a, wherein a is 3 or more nucleotides and wherein X 2  is at least 60% complementary to X 4 , wherein X 3  comprises a loop of the stem-loop, and wherein G x  comprises at least two consecutive G nucleotides;   wherein the stem-loop brings the label on the 5′-end-labeled nucleotide and the at least two consecutive G nucleotides into proximity, thereby quenching a detectable signal from the 5′-end-labeled nucleotide in the absence of a target nucleic acid sequence;   wherein the sequence-specific primer can hybridize to the target nucleic acid sequence;   wherein the PET tag does not substantially hybridize to the target nucleic acid sequence recognized by the sequence-specific primer; and   wherein the detectable signal from the 5′-end-labeled nucleotide is unquenched when the labeled sequence-specific primer is incorporated into an amplicon.   
     
     
         2 . The method of  claim 1 , wherein the PET tag comprises the sequence 5′-X 1 X 2(a) X 3 X 4(a) G x X 5(n) -3′ (SEQ ID NO: 2), wherein X 5(n)  comprises one or more nucleotides. 
     
     
         3 . The method of  claim 1 , wherein X 1  is not G. 
     
     
         4 . The method of  claim 1 , wherein X 2  has at least 80% complementarity to X 4 . 
     
     
         5 . The method of  claim 4 , wherein X 2  is 100% complementarity to X 4 . 
     
     
         6 . The method of  claim 1 , wherein X 3  is 3 or more nucleotides. 
     
     
         7 . The method of  claim 1 , wherein X 3  is a trinucleotide sequence selected from the group consisting of TAA, ATA, AAT, TTA, TAT, ATT, TTT and AAA. 
     
     
         8 . The method of  claim 1 , wherein X 3  does not include C or G nucleotides. 
     
     
         9 . The method of  claim 1 , wherein the label is a fluorophore. 
     
     
         10 . The method of  claim 1 , wherein the PET tag is 12 to 20 nucleotides in length. 
     
     
         11 . The method of  claim 1 , wherein a 5′-end of the sequence-specific primer is attached to the 3′-end of the PET tag. 
     
     
         12 . A labeled sequence-specific primer generated using the method of  claim 1 . 
     
     
         13 . A kit comprising:
 the labeled sequence-specific primer of  claim 12 ; and   a buffer.   
     
     
         14 . A method of detecting a target nucleic acid molecule comprising:
 incubating a sample comprising the target nucleic acid molecule with a forward primer comprising a sequence homologous to the target nucleic acid molecule and a reverse primer comprising a sequence homologous to the target nucleic acid molecule under conditions sufficient to allow amplification of the target nucleic acid molecule;   amplifying the target nucleic acid molecule using real-time polymerase chain reaction (PCR), thereby generating a labeled amplicon;   denaturing the labeled amplicon;   generating a melting curve; and   detecting a signal from the label,   wherein the forward primer or the reverse primer is linked at its 5′-end to the 3′-end of a photoinduced electron transfer (PET) tag, wherein the PET tag comprises the sequence 5′-X 1 X 2(a) X 3 X 4(a) G x -3′ (SEQ ID NO: 1), wherein X 1  is a 5′-end labeled nucleotide, wherein X 2  and X 4  comprise a stem of a stem-loop and are nucleotide sequences of length a, wherein a is 3 or more nucleotides and wherein X 2  is at least 60% complementary to X 4 , wherein X 3  comprises a loop of the stem-loop, and wherein G x  comprises at least two consecutive G nucleotides, wherein the stem-loop brings the label on the 5′-end-labeled nucleotide and the at least two consecutive G nucleotides into proximity, thereby quenching a detectable signal from the 5′-end-labeled nucleotide in the absence of a target nucleic acid sequence, and wherein the PET tag does not substantially hybridize to the target nucleic acid sequence recognized by the forward and reverse primers, and wherein the detectable signal from the 5′-end-labeled nucleotide is unquenched when the labeled forward or reverse primer is incorporated into the labeled amplicon,   wherein an increase in signal detected during the real-time PCR indicates that the target nucleic acid molecule is present in the sample and wherein no significant increase in signal detected during the real-time PCR indicates that the target molecule is not present in the sample, and   wherein a single peak detected during generating the melting curve indicates that the target nucleic acid molecule is present in the sample and wherein no single peak detected during generating the melting curve indicates that the target molecule is not present in the sample.   
     
     
         15 . The method of  claim 14 , further comprising quantifying the signal from the label. 
     
     
         16 . The method of  claim 14 , wherein the forward primer is linked at its 5′-end to the 3′-end portion of the PET tag and wherein the reverse primer is not linked to the PET tag. 
     
     
         17 . The method of  claim 14 , wherein the reverse primer is linked at its 5′-end to the 3′-end portion of the PET tag and wherein the forward primer is not linked to the PET tag. 
     
     
         18 . A method of detecting a polymorphism in a target nucleic acid molecule comprising:
 incubating a sample comprising the target nucleic acid molecule with a forward primer and a reverse primer, wherein the forward primer or the reverse primer is linked at its 5′-end to the 3′-end of a PET tag under conditions sufficient to allow amplification of the target nucleic acid molecule, thereby generating a labeled amplicon, wherein the PET tag comprises the sequence 5′-X 1 X 2(a) X 3 X 4(a) G x -3′ (SEQ ID NO: 1), wherein X 1  is a 5′-end labeled nucleotide, wherein X 2  and X 4  comprise a stem of a stem-loop and are nucleotide sequences of length a, wherein a is 3 or more nucleotides and wherein X 2  is at least 60% complementary to X 4 , wherein X 3  comprises a loop of the stem-loop, and wherein G x  comprises at least two consecutive G nucleotides, wherein the stem-loop brings the label on the 5′-end-labeled nucleotide and the at least two consecutive G nucleotides into proximity, thereby quenching a detectable signal from the 5′-end-labeled nucleotide in the absence of a target nucleic acid sequence, and wherein the PET nucleic acid sequence does not substantially hybridize to the target nucleic acid sequence recognized by the forward and reverse primers, and wherein the detectable signal from the 5′-end-labeled nucleotide is unquenched when the labeled forward or reverse primer is incorporated into an amplicon; and   detecting a change in signal from the label while exposing the labeled amplicon to conditions that permit denaturation of the amplicon into single-stranded nucleic acid molecules, wherein the change in signal is directly proportional to the extent of amplicon denaturation, and wherein differences in the extent of amplicon denaturation represent a polymorphism in the target nucleic acid.   
     
     
         19 . The method of  claim 18 , wherein the forward primer is linked at its 5′-end to the 3′-end of the PET tag and wherein the reverse primer is not linked to the PET tag. 
     
     
         20 . The method of  claim 18 , wherein the reverse primer is linked at its 5′-end to the 3′-end of the PET tag and wherein the forward primer is not linked to the PET tag.