Compositions and methods for analysis of nucleic acid molecules during amplification reactions
AssigneeHologic, Inc.
InventorsHatim T. Allawi, Victor I. Lyamichev
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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.
Claims31
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.
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 Tm with respect to said target nucleic acid that is at least 5° C. below the calculated Tm of at least one of said amplification primers with respect to said target nucleic acid.
Dependencies are inferred from the claim text.