Compositions and methods for analysis of nucleic acid molecules during amplification reactions

Abstract

The present invention provides systems, methods and kits for performing a detection assay (e.g., invasive cleavage assay) in combination with an amplification assay (e.g., PCR), where the detection assay employs enzyme footprint probes with relatively short (e.g., 6-12 bases) analyte-specific regions configured to provide a preferred footprint length of duplex for use with a particular nucleic acid modifying enzyme. In some embodiments, such assays are used for target quantification, and in other embodiments, such assays are used for genotyping. In certain embodiments, the use of such short probes allows for assays with increased dynamic range.

1 . A composition for analysis of a target nucleic acid, comprising: a) amplification primers; b) a polymerase; c) a FEN-1 endonuclease; and d) a synthetic probe comprising an analyte-specific portion and a non-target portion, wherein said non-target portion is substantially non-complementary to said target nucleic acid and wherein said analyte-specific portion is no more than 12 nucleotides in length, and contains up to 12 nucleotides that are complementary to said target nucleic acid. 
     
     
         2 . The composition of  claim 1 , wherein said analyte specific portion of said synthetic probe has a calculated T m  with respect to said target nucleic acid at least 5° C. below the calculated T m  of at least one of said amplification primers with respect to said target nucleic acid. 
     
     
         3 . The composition of  claim 2 , wherein said analyte specific portion of said synthetic probe has a calculated T m  with respect to said target nucleic acid at least 8° C. below the calculated T m  of at least one of said amplification primers with respect to said target nucleic acid. 
     
     
         4 . The composition of  claim 3 , wherein said analyte specific portion of said synthetic probe has a calculated T m  with respect to said target nucleic acid at least 10° C. below the calculated T m  of at least one of said amplification primers with respect to said target nucleic acid. 
     
     
         5 . The composition of  claim 1 , further comprising a reverse transcriptase enzyme. 
     
     
         6 . The composition of  claim 1 , wherein said polymerase is a thermostable DNA polymerase. 
     
     
         7 . The composition of  claim 6 , wherein said thermostable DNA polymerase lack 5′ to 3′ exonuclease activity. 
     
     
         8 . The composition of  claim 1 , wherein said FEN-1 endonuclease is a thermostable FEN-1 endonuclease. 
     
     
         9 . The composition of  claim 1 , wherein said FEN-1 endonuclease is from an archaeal species. 
     
     
         10 . The composition of  claim 1 , further comprising a second oligonucleotide configured to form a cleavage structure with said probe in the presence of said target nucleic acid, wherein said cleavage structure is formed by association of said target nucleic acid with: a) said probe hybridized at a first region of said target nucleic acid; and b) said second oligonucleotide hybridized at a second region of said target nucleic, wherein said second region is downstream of said first region. 
     
     
         11 . The composition of  claim 10 , wherein said second region of said target nucleic acid is contiguous with and downstream of said first region of said target nucleic acid. 
     
     
         12 . The composition of  claim 11 , wherein at least one nucleotide at the 3′ end of the second oligonucleotide, when associated with said target in a cleavage structure, overlaps with a region of hybridization between said probe and said target nucleic acid. 
     
     
         13 . The composition of  claim 12 , wherein the 3′ terminal nucleotide of said second oligonucleotide is not complementary to said target nucleic acid, when said second oligonucleotide is associated with said target nucleic acid in said cleavage structure. 
     
     
         14 . The composition of  claim 1 , wherein said synthetic probe is unlabeled. 
     
     
         15 . The composition of  claim 1 , wherein said synthetic probe does not contain non-natural nucleotides. 
     
     
         16 . The composition of  claim 1 , wherein said synthetic probe does not comprise a minor groove binder moiety. 
     
     
         17 . The composition of  claim 1 , further comprising a synthetic detection oligonucleotide having a region complementary to said non-target portion of said synthetic probe. 
     
     
         18 . The composition of  claim 17 , wherein said synthetic detection oligonucleotide has a region of self-complementarity that forms a hairpin structure. 
     
     
         19 . The composition of  claim 17 , wherein said synthetic detection oligonucleotide comprises a label. 
     
     
         20 . The composition of  claim 19 , wherein said label is a fluorescent label. 
     
     
         21 . The composition of  claim 20 , wherein said synthetic detection oligonucleotide further comprises a fluorescence quencher moiety. 
     
     
         22 . The composition of  claim 1 , further comprising a target nucleic acid. 
     
     
         23 . A composition comprising: a) a target nucleic acid; b) amplification primers; c) a polymerase; d) a FEN-1 endonuclease; and e) a synthetic probe comprising an analyte-specific portion and a non-target portion, wherein said non-target portion is substantially non-complementary to said target nucleic acid, and wherein said analyte-specific portion of said synthetic probe has a calculated T m  with respect to said target nucleic acid that is at least 5° C. below the calculated T m  of at least one of said amplification primers with respect to said target nucleic acid. 
     
     
         24 . The composition of  claim 23 , wherein said wherein said analyte-specific portion of said unlabeled synthetic probe has a calculated T m  with respect to said target nucleic acid that is at least 8° C. below the calculated T m  of at least one of said amplification primers with respect to said target nucleic acid. 
     
     
         25 . The composition of  claim 23 , wherein said wherein said analyte-specific portion of said unlabeled synthetic probe has a calculated T m  with respect to said target nucleic acid that is at least 10° C. below the calculated T m  of at least one of said amplification primers with respect to said target nucleic acid. 
     
     
         26 . The composition of  claim 23 , further comprising a synthetic detection oligonucleotide having a region complementary to said non-target portion of said synthetic probe. 
     
     
         27 . The composition of  claim 26 , wherein said synthetic detection oligonucleotide has a region of self-complementarity that forms a hairpin structure. 
     
     
         28 . The composition of  claim 26 , wherein said synthetic detection oligonucleotide comprises a label. 
     
     
         29 . The composition of  claim 28 , wherein said label is a fluorescent label. 
     
     
         30 . The composition of  claim 29 , wherein said synthetic detection oligonucleotide further comprises a fluorescence quencher moiety. 
     
     
         31 . The composition of  claim 23 , further comprising a second oligonucleotide configured to form a cleavage structure with said probe in the presence of said target nucleic acid, wherein said cleavage structure is formed by association of said target nucleic acid with: a) said synthetic probe hybridized at a first region of said target nucleic acid; and b) said second oligonucleotide hybridized at a second region of said target nucleic, wherein said second region is downstream of said first region.