Compositions and methods for the detection of nucleic acids

Abstract

The present invention provides methods for detecting a target nucleic acid in a sample by, for example, incubating the target nucleic acid with a detection probe containing a nucleic acid sequence complementary to at least a portion of the target nucleic acid and a nuclease enzyme that specifically cleaves double-stranded nucleic acids. Hybridization between the detection probe and the target nucleic acid thereby leads to cleavage of the detection probe, releasing a portion of the probe attached to a detectable agent. The portions of the digested probes attached to the detectable agent can be separated from unbound probe and detected in order to determine the presence of the target nucleic acid in the sample. Thus, the invention enables rapid and accurate analysis of a sample for the presence of desired nucleic acid biomarkers.

What is claimed is: 
     
         1 . A composition comprising:
 (a) a clinical specimen comprising a target nucleic acid,   (b) a nucleic acid probe,   (c) a lysis buffer, and   (d) a duplex-specific nuclease (DSN).   
     
     
         2 . The composition of  claim 1 , wherein said clinical specimen comprises a blood specimen, a buccal specimen, a nasal specimen, a fecal specimen, a tissue specimen, a urine specimen, or a bacterial specimen. 
     
     
         3 . (canceled) 
     
     
         4 . The composition of  claim 1 , wherein said lysis buffer comprises 0.1-2% sodium dodecyl sulfate (SDS). 
     
     
         5 . The composition of  claim 4 , wherein said lysis buffer comprises 1% SDS %. 
     
     
         6 . The composition of  claim 1 , wherein said lysis buffer comprises proteinase K. 
     
     
         7 . The composition of  claim 1 , wherein said DSN is a Kamchatka crab DSN. 
     
     
         8 . The composition of  claim 1 , wherein said nucleic acid probe is attached to a surface. 
     
     
         9 .- 70 . (canceled) 
     
     
         71 . The composition of  claim 1 , wherein said clinical specimen has not undergone purification or amplification. 
     
     
         72 . The composition of  claim 1 , wherein said nucleic acid probe is a hairpin probe comprising a first region, a second region, and a third region, wherein said first region is complementary to said third region. 
     
     
         73 . The composition of  claim 72 , wherein said second region of said nucleic acid probe is complementary to a portion of said target nucleic acid. 
     
     
         74 . The composition of  claim 1 , wherein said target nucleic acid is RNA. 
     
     
         75 . The composition of  claim 1 , wherein said target nucleic acid is DNA. 
     
     
         76 . The composition of  claim 1  further comprising a detection probe. 
     
     
         77 . The composition of  claim 76 , wherein said detection probe comprises a nucleic acid region that binds to the first region or the third region of said nucleic acid probe. 
     
     
         78 . The composition of  claim 76 , wherein said detection probe is attached to a detectable label. 
     
     
         79 . The composition of  claim 78 , wherein said detectable label is a fluorophore. 
     
     
         80 . The composition of  claim 79 , wherein said detection probe is further attached to a quencher. 
     
     
         81 . The composition of  claim 80 , wherein said fluorophore is attached to one end of said detection probe and said quencher is attached to the opposite end of said detection probe.