Primer for nucleic acid detection

Abstract

This application provides universal labeled primers for detection and amplification of nucleic acid molecules. These universal primers can be attached to the 5′-end of a target sequence-specific primer. In particular examples, the universal primer includes a labeled nucleotide flanked on both sides a nucleotide whose complement nucleotides changes a detectable signal from the label when the universal primer hybridizes with its complementary nucleic acid molecule. Also disclosed are methods of using the universal primer in nucleic acid amplification, such as real-time PCR.

1 . An isolated nucleic acid molecule comprising: 
 a 5′ end;    a 3′ end; and    a label on a nucleotide, wherein the labeled nucleotide is flanked by two nucleotides whose complement nucleotides change a detectable signal from the label when said isolated nucleic acid molecule is hybridized with its complementary nucleic acid molecule.    
     
     
         2 . The isolated nucleic acid molecule of  claim 1 , wherein the isolated molecule comprises the sequence 5′-X 1(n) X 2 X 3 X 4 X 5(n) -3′, wherein X 3  is the labeled nucleotide, wherein X 2  and X 4  are the two nucleotides flanking the labeled nucleotide, and wherein n is zero or more nucleotides.  
     
     
         3 . The isolated nucleic acid molecule of  claim 1 , wherein the isolated molecule comprises the sequence 5′-X 1(n) X 2 X 3 X 4 X 5(n) -3′, wherein X 3  is the labeled nucleotide, wherein X 2  and X 4  are the two nucleotides flanking the labeled nucleotide, and wherein n is one or more nucleotides.  
     
     
         4 .- 5 . (canceled)  
     
     
         6 . The isolated nucleic acid molecule of  claim 2 , wherein the isolated molecule comprises the sequence 5′-X 2 X 3 X 4 X 5(n) -3′, wherein X 3  is the labeled nucleotide, wherein X 2  and X 4  are the two nucleotides flanking the labeled nucleotide, and wherein n is one or more nucleotides.  
     
     
         7 . The isolated nucleic acid molecule of  claim 6 , wherein the isolated molecule comprises the sequence 5′-X 2 X 3 X 4 X 5(n) -3′, wherein X 2  and X 4  are “C”.  
     
     
         8 . The isolated nucleic acid molecule of  claim 6 , wherein the isolated molecule comprises the sequence 5′-X 2 X 3 X 4 X 5(n) -3′, wherein X 3  is “T”.  
     
     
         9 . The isolated nucleic acid molecule of  claim 6 , wherein the isolated molecule comprises the sequence 5′-X 2 X 3 X 4 X 5(n) -3′, wherein X 3  is “T” and wherein X 2  and X 4  are “C”.  
     
     
         10 . The isolated nucleic acid molecule of  claim 3  wherein the isolated molecule comprises the sequence 5′-CTCSX (n) -3 (SEQ ID NO: 20), wherein X (n)  is any number of nucleotides, wherein S is G or C, and wherein T is the labeled nucleotide.  
     
     
         11 .- 16 . (canceled)  
     
     
         17 . The isolated nucleic acid molecule of  claim 10 , wherein the isolated nucleic acid molecule comprises the sequence 5′-CTCSXXX-3′ (SEQ ID NO: 21), wherein X is any nucleotide, wherein S is G or C, and wherein T is the labeled nucleotide.  
     
     
         18 . The isolated nucleic acid molecule of  claim 10 , wherein the isolated nucleic acid molecule comprises the nucleic acid sequence shown in SEQ ID NO: 1, 6, 8, 14, 18, 19, 21, 22, 29,31, or 32.  
     
     
         19 . An isolated nucleic acid molecule comprising 5′-CTCSXXX (n) -3′ (SEQ ID NO:  22 ), wherein a fluorophore is attached to “T” and n comprises 0-11 nucleotides.  
     
     
         20 .- 28 . (canceled)  
     
     
         29 . The isolated nucleic acid molecule of  claim 10 , wherein the isolated nucleic acid molecule comprises a target-specific probe sequence.  
     
     
         30 . The isolated nucleic acid molecule of  claim 29 , wherein the isolated nucleic acid molecule comprises the sequence 5′-CTCSSSSX (n) RRRRGAG-3′ (SEQ ID NO: 31), wherein X (n)  is any number of nucleotides comprising a target-specific probe, wherein S is G or C, wherein R is the complementary nucleotide to S, and wherein T is the labeled nucleotide.  
     
     
         31 . The isolated nucleic acid molecule of  claim 30 , wherein the isolated nucleic acid molecule comprises the sequence 5′-CTCSSSSX (n) RRRRGAG(Z (n) )X (n) -3′ (SEQ ID NO: 32), wherein X (n)  is any number of nucleotides comprising a target-specific nucleotide sequence, wherein Z(n) is a carbon spacer arm and n is a spacer phophoramidite containing at least 3 carbons.  
     
     
         32 . The isolated nucleic acid molecule of  claim 31 , wherein Z (n)  is a blocking moiety that can reduce or prevent copying of the 5′ tail sequence.  
     
     
         33 . The nucleic acid molecule of  claim 31 , wherein the fluorophore signal is emitted upon hybridization of the probe to the target nucleic acid in the primer extension product.  
     
     
         34 . A kit comprising: 
 the isolated nucleic acid molecule of  claim 1;  and    a ligase.    
     
     
         35 . An array comprising the isolated nucleic acid molecule of  claim 1 .  
     
     
         36 . A method of detecting 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 the isolated nucleic acid molecule of  claim 1  under conditions sufficient to allow amplification of the target nucleic acid molecule, thereby generating a labeled amplicon; and    detecting a change in signal from the label during or after amplification relative to signal from the label before the amplification, wherein a change in signal indicates that the target nucleic acid molecule is present in the sample and wherein no significant change in signal indicates that the target nucleic acid molecule is not present in the sample.    
     
     
         37 . (canceled)  
     
     
         38 . The method of  claim 36 , further comprising: 
 incubating the labeled amplicon with an array comprising the target nucleic acid molecule under conditions that permit hybridization between the labeled amplicon and the target nucleic acid; and    detecting a signal from the fluorophore, wherein an increase in signal indicates that the target nucleic acid molecule is present in the sample and wherein no significant increase in signal indicates that the target nucleic acid molecule is not present in the sample.    
     
     
         39 .- 46 . (canceled)  
     
     
         47 . A method of detecting 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 the isolated nucleic acid molecule of  claim 1  under conditions sufficient to allow amplification of the target nucleic acid molecule, thereby generating a labeled amplicon;    exposing the labeled amplicon to conditions that permit denaturation of the amplicon into single strand nucleic acid molecules;    exposing the single strand nucleic acid molecules to conditions that permit annealing between the single strand nucleic acid molecules and the forward primer or the reverse primer linked at its 5′ end to the 3′ end of the isolated nucleic acid molecule of  claim 1;  and    detecting a change in signal from the label during denaturation relative to signal from the label before the denaturation, wherein a change in signal indicates that the target nucleic acid molecule is present in the sample and wherein no significant change in signal indicates that the target nucleic acid molecule is not present in the sample.    
     
     
         48 . A method of detecting 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 the isolated nucleic acid molecule of  claim 31  under conditions sufficient to allow amplification of the target nucleic acid molecule, thereby generating a labeled amplicon; and    detecting a change in signal from the label during or after amplification relative to signal from the label before the amplification, wherein a change in signal indicates that the target nucleic acid molecule is present in the sample and wherein no significant change in signal indicates that the target nucleic acid molecule is not present in the sample.    
     
     
         49 . (canceled)