Polynucleotide primers and probes

Abstract

The present invention provides a novel technology that involves improved primer design. These primer pairs have a wide range of applications and provide high sensitivity and specificity.

1 . A polynucleotide primer combination comprising a first polynucleotide and a second polynucleotide,
 the first polynucleotide (P) comprising a first domain (Pa) having a sequence that is complementary to a first target polynucleotide region (TO and a second domain (Pc) comprising a unique polynucleotide sequence, and   the second polynucleotide (F) comprising a first domain (Fb) having a sequence that is complementary to a second target polynucleotide region (T 2 ) and a second domain (Fd) comprising a polynucleotide sequence sufficiently complementary to Pc such that Pc and Fd will hybridize under appropriate conditions,   wherein the target polynucleotide has a secondary structure that is denatured by hybridization of Fb to the target polynucleotide.   
     
     
         2 . The polynucleotide primer combination of  claim 1  wherein the secondary structure of the target polynucleotide inhibits polymerase extension of the target polynucleotide in the absence of F. 
     
     
         3 . The polynucleotide primer combination of  claim 1  wherein the P and/or F further comprise a modified nucleic acid. 
     
     
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         5 . A polynucleotide primer combination comprising a first polynucleotide, a second polynucleotide, and a blocker polynucleotide,
 the first polynucleotide (P) comprising a first domain (Pa) having a sequence that is complementary to a first target polynucleotide region (TO and a second domain (Pc) comprising a unique polynucleotide sequence,   the second polynucleotide (F) comprising a first domain (Fb) having a sequence that is complementary to a second target polynucleotide region (T 2 ) and a second domain (Fd) comprising a polynucleotide sequence sufficiently complementary to Pc such that Pc and Fd will hybridize under appropriate conditions, and   the blocker polynucleotide comprising a nucleotide sequence that is complementary to a third target polynucleotide region (T 3 ), wherein T 3  is located 5′ of T 1  and T 2 .   
     
     
         6 . The polynucleotide primer combination of  claim 5  wherein a nucleotide at the 3′ end of P and a nucleotide at the 5′ end of the blocker polynucleotide overlap. 
     
     
         7 . The polynucleotide primer combination of  claim 6  wherein the blocker polynucleotide has a sequence that overlaps Pa over the whole length of Pa. 
     
     
         8 . The polynucleotide primer combination of  claim 6  wherein the nucleotide at the 3′ end of P and the nucleotide at the 5′ end of the blocker polynucleotide are different. 
     
     
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         32 . The polynucleotide primer combination of  claim 1  wherein Pa is from about 5 bases in length to about 30 bases in length, about 5 bases in length to about 20 bases in length, about 5 bases in length to about 15 bases in length, about 5 bases in length to about 10 bases in length, about 5 bases in length to about 8 bases in length. 
     
     
         33 . The polynucleotide primer combination of  claim 1  wherein Pc is from about 5 bases in length to about 200 bases in length, about 5 bases in length to about 150 bases in length, about 5 bases in length to about 100 bases in length, about 5 bases in length to about 50 bases in length, about 5 bases in length to about 45 bases in length, about 5 bases in length to about 40 bases in length, about 5 bases in length to about 35 bases in length, about 5 bases in length to about 30 bases in length, about 5 bases in length to about 25 bases in length, about 5 bases in length to about 20 bases in length, about 5 bases in length to about 15 bases in length, about 10 to about 50 bases in length, about 10 bases in length to about 45 bases in length, about 10 bases in length to about 40 bases in length, about 10 bases in length to about 35 bases in length, about 10 bases in length to about 30 bases in length, about 10 bases in length to about 25 bases in length, about 10 bases in length to about 20 bases in length, or about 10 bases in length to about 15 bases in length. 
     
     
         34 . The polynucleotide primer combination of  claim 1  wherein Fb is from about 10 bases in length to about 5000 bases in length, about 10 bases in length to about 4000 bases in length, about 10 bases in length to about 3000 bases in length, about 10 bases in length to about 2000 bases in length, about 10 bases in length to about 1000 bases in length, about 10 bases in length to about 500 bases in length, about 10 bases in length to about 250 bases in length, about 10 bases in length to about 200 bases in length, about 10 bases in length to about 150 bases in length, about 10 bases in length to about 100 bases in length, about 10 bases in length to about 95 bases in length, about 10 bases in length to about 90 bases in length, about 10 bases in length to about 85 bases in length, about 10 bases in length to about 80 bases in length, about 10 bases in length to about 75 bases in length, about 10 bases in length to about 70 bases in length, about 10 bases in length to about 65 bases in length, about 10 bases in length to about 60 bases in length, about 10 bases in length to about 55 bases in length, about 10 bases in length to about 50 bases in length, about 10 bases in length to about 45 bases in length, about 10 bases in length to about 40 bases in length, about 10 bases in length to about 35 bases in length, about 10 bases in length to about 30 bases in length, or about 10 bases in length to about 100 bases in length. 
     
     
         35 . The polynucleotide primer combination of  claim 1  wherein Fd is from about 5 bases in length to about 200 bases in length, about 5 bases in length to about 150 bases in length, about 5 bases in length to about 100 bases in length, about 5 bases in length to about 50 bases in length, about 5 bases in length to about 45 bases in length, about 5 bases in length to about 40 bases in length, about 5 bases in length to about 35 bases in length, about 5 bases in length to about 30 bases in length, about 5 bases in length to about 25 bases in length, about 5 bases in length to about 20 bases in length, about 5 bases in length to about 15 bases in length, about 10 to about 50 bases in length, about 10 bases in length to about 45 bases in length, about 10 bases in length to about 40 bases in length, about 10 bases in length to about 35 bases in length, about 10 bases in length to about 30 bases in length, about 10 bases in length to about 25 bases in length, about 10 bases in length to about 20 bases in length, or about 10 bases in length to about 15 bases in length. 
     
     
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         45 . A method of detecting the presence of a target polynucleotide in a sample with a primer combination, the primer combination comprising a first polynucleotide and a second polynucleotide,
 the first polynucleotide (P) comprising a first domain (Pa) having a sequence that is fully complementary to a first target polynucleotide region (T 1 ) and a second domain (Pc) comprising a unique polynucleotide sequence, Pa having a sequence that is not fully complementary to a non-target polynucleotide in the sample and   the second polynucleotide (F) comprising a first domain (Fb) that is complementary to a second target polynucleotide region (T 2 ) and a second domain (Fd) comprising a polynucleotide sequence sufficiently complementary to Pc such that Pc and Fd will hybridize under appropriate conditions,   the method comprising the steps of:   contacting the sample with the primer combination and a polymerase under conditions that allow extension of a sequence from Pa which is complementary to the target polynucleotide when the target polynucleotide is present in the sample and   detecting the sequence extended from Pa indicating the presence of the target polynucleotide in the sample.   
     
     
         46 . The method of  claim 45  wherein the method provides a change in sequence detection from a sample with a non-target polynucleotide compared to sequence detection from a sample with a target polynucleotide. 
     
     
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         52 . The method of  claim 45  wherein detection is carried out in real time. 
     
     
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