Cleavable competitor polynucleotides

Abstract

The invention relates to polynucleotide combinations and their use in allele-specific enrichment, amplification, and detection. The disclosure also provides methods to multiplex various target DNA molecules in a single tube with high sensitivity and specificity. The disclosure provides a polynucleotide competitor that comprises a sequence that is fully complementary to a first target DNA polynucleotide region (T1) such that the competitor polynucleotide will hybridize to the first target DNA polynucleotide region under appropriate conditions. In another aspect, the polynucleotide competitor comprises a mismatch to a non-target DNA polynucleotide that is a sequence variant of the first target DNA polynucleotide region (T1*).

What is claimed is: 
     
       1. A composition comprising a first polynucleotide and a second polynucleotide, wherein: (A) the first polynucleotide comprises a sequence such that: (i) the first polynucleotide is fully complementary to a target polynucleotide region (T1) such that the first polynucleotide is able to hybridize to T1 under appropriate conditions, and the sequence comprises a RNA base that is susceptible to cleavage by a ribonuclease when the RNA base is hybridized to T1; and (ii) the first polynucleotide is mismatched to a non-target polynucleotide region (T1*) at the position of the RNA base or 1, 2 or 3 nucleotides adjacent to the RNA base; and (iii) T1* is a sequence variant of T1; and (B) the second polynucleotide comprises a sequence such that: (iv) the second polynucleotide is fully complementary to a target polynucleotide region (T2) and a non-target polynucleotide region (T2) that overlaps T1 and T1* by at least one nucleotide, wherein T2 is upstream of T1 and T1*. 
     
     
       2. The composition of  claim 1 , wherein the RNA base on the first polynucleotide is located at the midpoint of the first polynucleotide. 
     
     
       3. The composition of  claim 1 , further comprising at least one additional RNA base on the first polynucleotide located immediately adjacent to the first RNA base. 
     
     
       4. The composition of  claim 3  wherein the first polynucleotide comprises at least 4 consecutive RNA bases. 
     
     
       5. The composition of  claim 4  wherein one or more RNA bases are susceptible to cleavage by a ribonuclease when the polynucleotide is hybridized to the target sequence T1. 
     
     
       6. The composition of  claim 5  wherein modified nucleotides at one or more RNA bases renders the one or more bases resistant to cleavage by a ribonuclease. 
     
     
       7. The composition of  claim 1  wherein the 3′ terminus of the first polynucleotide is blocked from initiation of extension by a DNA polymerase. 
     
     
       8. The composition of  claim 1  wherein the first polynucleotide comprises a detectable marker and a moiety that quenches the detectable marker. 
     
     
       9. The composition of  claim 8  wherein the detectable marker and the moiety are on opposite sides of the RNA base, and in a configuration that prevents detection of the detectable marker. 
     
     
       10. The composition of  claim 9  wherein cleavage of the first polynucleotide results in detection of the detectable marker. 
     
     
       11. The composition of  claim 1  wherein T2 overlaps T1 and T1* by at least about 1 to at least about 50 nucleotides. 
     
     
       12. The composition of  claim 1  wherein the ribonuclease includes but is not limited to RNase H2 or RNase H1. 
     
     
       13. A method of initiating polymerase extension on a target polynucleotide in a sample using the composition of  claim 1 ; wherein the sample comprises a target polynucleotide that comprises (i) a sequence T1 in a first region that is fully complementary to the sequence of a domain in the first polynucleotide; and (ii) a sequence T2 that is fully complementary to the sequence in the second polynucleotide; the method comprising the step of (a) contacting the sample with the composition and a polymerase under conditions that allow extension of a sequence from T2 following cleavage and dissociation of the first polynucleotide. 
     
     
       14. A method of amplifying a target polynucleotide in a sample using the composition of  claim 1 , wherein: the sample comprises a mixture of (i) a target polynucleotide having a sequence in a first region (T1) that is fully complementary to the sequence of a domain in the first polynucleotide, and a sequence in a second region (T2) that is fully complementary to the sequence in the second polynucleotide; and (ii) one or more non-target polynucleotides that are not fully complementary to T1; the method comprising the steps of: (a) contacting the sample with the composition and a polymerase under conditions that allow extension of a sequence (S) from T2, wherein the sequence is complementary to the target polynucleotide when the target polynucleotide is present in the sample; (b) denaturing the sequence (S) extended from T2 from the target polynucleotide, and (c) repeating step (a) in the presence of a third polynucleotide having a sequence complementary to a region (T3) in the sequence extended from T2 in step (b) to amplify the target polynucleotide, wherein extension and amplification of the target polynucleotide to generate a product occurs when the first polynucleotide is fully complementary to the sequence in T1, but is less efficient or does not occur when the first polynucleotide is not fully complementary to the sequence in a non-target sequence T1*; and wherein steps (a)-(c) are followed by further extension and amplification of the product when the first polynucleotide hybridizes to T1 or the second polynucleotide hybridizes to T2, and the third polynucleotide hybridizes to T3 in the presence of the polymerase. 
     
     
       15. The method of  claim 14 , further comprising: (iii) a fourth polynucleotide having a sequence that is fully complementary to a region T4 in a second target polynucleotide in the sample, such that the fourth polynucleotide is able to hybridize to T4 under appropriate conditions, and the sequence comprises a RNA base that is susceptible to cleavage by a ribonuclease when the RNA base is hybridized to T4; and (iv) a fifth polynucleotide having a sequence that is fully complementary to a region T5 in a second target polynucleotide in the sample, wherein T5 overlaps T4 and T4* by at least one nucleotide, and wherein T5 is upstream of T4 and T4*; wherein (v) the fourth polynucleotide is mismatched to a non-target polynucleotide region (T4*) at the position of the RNA base or 1, 2 or 3 bases adjacent to the RNA base; and wherein the sample comprises a mixture of (i) a target polynucleotide having a sequence in a first region (T4) that is fully complementary to the sequence of a domain in the fourth polynucleotide, and a sequence in a second region (T5) that is fully complementary to the sequence in the fifth polynucleotide; and (ii) one or more non-target polynucleotides that are not fully complementary to T4. 
     
     
       16. The method of  claim 13  further comprising the step of adding a ribonuclease at step (a). 
     
     
       17. The method of  claim 14  further comprising the step of adding a ribonuclease at step (a).