Primers and methods for the detection and discrimination of nucleic acids

Abstract

The present invention provides novel primers and methods for the detection of specific nucleic acid sequences. The primers and methods of the invention are useful in a wide variety of molecular biology applications and are particularly useful in allele specific PCR.

1 . A composition for quantifying or detecting one or more target nucleic acid molecules in a sample comprising one or more detectably labeled oligonucleotides and one or more target nucleic acid molecules to be detected or quantified, wherein said oligonucleotides comprise one of more detectable labels located internally and/or at or near the 3′ and/or 5′ termini of said oligonucleotides and wherein said label undergoes a detectable change in an observable property upon becoming part of a double stranded molecule. 
     
     
         2 . The composition of  claim 1 , wherein said detectable change is an increase or enhancement in the level of activity of the detectable label compared to the level of activity of the detectable label in the absence of said target nucleic acid molecules. 
     
     
         3 . The composition of  claim 2 , wherein said detectable labels are selected from the group consisting of fluorescent labels, chemiluminescent labels and bioluminescent labels. 
     
     
         4 . The composition of  claim 3 , wherein the fluorescent label is selected from the group consisting of FAM, TAMRA, JOE, Rhodamine, BODIPY, R6G, ROX, and EDANS. 
     
     
         5 . The composition of  claim 1 , wherein said one or more detectable labels are the same or different. 
     
     
         6 . The composition of  claim 1 , wherein one or more of said oligonucleotides comprise one or more hairpin structures. 
     
     
         7 . The composition of  claim 1 , wherein one or more of said oligonucleotides is hybridized to one or more of said nucleic acid molecules. 
     
     
         8 . The composition of  claim 1 , further comprising at least one component selected from the group consisting of one or more nucleotides, one or more DNA polymerases and one or more reverse transcriptases. 
     
     
         9 . The composition of  claim 1 , wherein said nucleic acid molecules are RNA and/or DNA molecules. 
     
     
         10 . A method for quantification or detection of one or more target nucleic acid molecules in a sample comprising hybridizing one or more detectably labeled oligonucleotides of  claim 1  with one or more molecules to be detected or quantified, and detecting the presence or absence and/or quantifying the amount of said target nucleic acid molecules. 
     
     
         11 - 17 . (canceled) 
     
     
         18 . A method for amplifying a double stranded nucleic acid molecule, comprising:
 providing a first and second primer, wherein said first primer is complementary to a sequence within or at or near the 3′-termini of the first strand of said nucleic molecule and said second primer is complementary to a sequence within or at or near the 3′-termini of the second strand of said nucleic acid molecule;   hybridizing said first primer to said first strand and said second primer to said second strand in the presence of one or more of the polymerases, under conditions such that a third nucleic acid molecule complementary to all or a portion of said first strand and a fourth nucleic acid molecule complementary to all or a portion said second strand are synthesized;   denaturing said first and third strand, and said second and fourth strands; and   repeating the above steps one or more times, wherein one or more of the primers comprise a detectable label internally and/or at or near its 3′ and/or 5′ termini and/or comprises one or more hairpin structures.   
     
     
         19 . The method of  claim 18 , wherein at least one of said primers comprises at least one hairpin structure. 
     
     
         20 - 47 . (canceled) 
     
     
         48 . A method of determining the presence of at least one particular nucleotide of interest at a specific position in a target nucleic acid molecule, comprising:
 providing at least one target nucleic acid molecule having said nucleotide of interest at a specific position;   contacting said target nucleic acid molecule with at least one oligonucleotide, wherein at least a portion of the oligonucleotide is capable of forming base pairs or hybridizing with at least a portion of the nucleic acid molecule and wherein the oligonucleotide comprises at least one specificity enhancing group and/or at least one label; and   contacting the oligonucleotide and the target nucleic acid molecule with a polymerase less able to extend the oligonucleotide when the 3′-most nucleotide of the oligonucleotide does not base pair with the target nucleic acid and more able to extend the oligonucleotide when the 3′-most nucleotide of the oligonucleotide base pairs with the target nucleic acid molecule.   
     
     
         49 . The method of  claim 48 , wherein the polymerase enzyme is Tsp DNA polymerase. 
     
     
         50 . The method of  claim 48 , wherein the group is a fluorescent moiety. 
     
     
         51 . The method according to  claim 48 , wherein the groups is attached to a nucleotide at or near the 3′-nucleotide. 
     
     
         52 . The method according to  claim 48 , wherein the group is attached to one of the ten 3′-most nucleotides. 
     
     
         53 . The method according to  claim 48 , wherein the group is detectable. 
     
     
         54 . The method according to  claim 48 , wherein the oligonucleotide is in the form of a hairpin. 
     
     
         55 . (canceled)