Methods and compositions for detecting target nucleic acids

Abstract

The present invention provides compositions, apparatuses and methods for detecting one or more nucleic acid targets present in a sample. Methods of the invention include utilizing two or more ligation probes that reversibly bind a target nucleic acid in close proximity to each other and possess complementary reactive ligation moieties. When such probes have bound to the target in the proper orientation, they are able to undergo a spontaneous chemical ligation reaction that yields a ligation product that is directly detected or that is amplified to produce amplicons that are then detected. The present invention also provides methods to stabilize sample RNA so that degradation does not significantly affect the results of the analysis.

1 .- 32 . (canceled) 
     
     
         33 . A method for detecting a plurality of different target nucleic acids in a blood sample, wherein each target nucleic acid comprises a first target domain adjacent to a second target domain, said method comprising:
 (a) contacting the blood sample comprising the different target nucleic acids with a lysis buffer to form a reaction mixture;   (b) contacting the reaction mixture directly with a plurality of different probe sets, each probe set comprising:
 (i) a first ligation probe comprising:
 (1) a first probe domain complementary to the first target domain of one target nucleic acid in the plurality of target nucleic acids; 
 (2) a first primer sequence; and 
 (3) a 5′ ligation moiety; and 
 
 (ii) a second ligation probe comprising:
 (1) a second probe domain complementary to the second target domain of the one target nucleic acid in the plurality of target nucleic acid; 
 (2) a second primer sequence; and 
 (3) a 3′ ligation moiety; 
 
   (c) ligating said first and second ligation probes without the use of a ligase enzyme to form a plurality of different ligation products;   (d) amplifying the ligation different ligation products; and   (e) detecting the presence of the ligation products   
     
     
         34 . A method for detecting a plurality of different target nucleic acids in a formalin-fixed, paraffin-embedded (FFPE) tissue sample, wherein each target nucleic acid comprises a first target domain adjacent to a second target domain, said method comprising:
 (a) contacting the FFPE tissue sample comprising the different target nucleic acids with a lysis buffer to form a reaction mixture;   (b) contacting the reaction mixture directly with a plurality of different probe sets, each probe set comprising:
 (i) a first ligation probe comprising:
 (1) a first probe domain complementary to the first target domain of one target nucleic acid in the plurality of target nucleic acids; 
 (2) a first primer sequence; and 
 (3) a 5′ ligation moiety; and 
 
 (ii) a second ligation probe comprising:
 (1) a second probe domain complementary to the second target domain of the one target nucleic acid in the plurality of target nucleic acid; 
 (2) a second primer sequence; and 
 (3) a 3′ ligation moiety; 
 
   (c) ligating said first and second ligation probes without the use of a ligase enzyme to form a plurality of different ligation products;   (d) amplifying the different ligation products to form amplicons; and   (e) detecting the amplicons.   
     
     
         35 . The method of  claim 33 , wherein step (a) and (b) are performed at the same location. 
     
     
         36 . The method of  claim 33 , wherein step (a) and (b) are performed at different locations. 
     
     
         37 . The method of  claim 33 , wherein step (b) is performed one day to three months after step (a). 
     
     
         38 . The method of  claim 33 , wherein each of the ligation probes comprises a primer sequence. 
     
     
         39 . The method of  claim 33 , wherein the 5′ ligation moiety comprises DABSYL and the 3′ ligation moiety comprises phosphorothioate. 
     
     
         40 . The method of  claim 33 , wherein the lysis buffer comprises gunadinium hydrochloride (GuHCl). 
     
     
         41 . The method of  claim 40 , wherein the lysis buffer comprises 3M GuHCl. 
     
     
         42 . The method of  claim 33 , wherein the target nucleic acids are DNA. 
     
     
         43 . The method of  claim 33 , wherein the target nucleic acids are RNA. 
     
     
         44 . A method for assessing the degradation of a target nucleic acid in a sample, wherein the target nucleic acid comprises a first target domain adjacent to a second target domain, a third target domain adjacent to a fourth target domain and a target capture domain,
 wherein the third target domain and fourth target domain are upstream of the first target domain and second target domain,   wherein the first target capture domain is downstream of the first target domain and second target domain, and   wherein the first, second, third and fourth target domains are at known distances from the target capture probe domain,   the method comprising:   (a) contacting the sample comprising the target nucleic acid with a lysis buffer to form a reaction mixture;   (b) contacting the reaction mixture with a probe set, the probe set comprising:
 (i) a first ligation probe comprising:
 (1) a first probe domain complementary to the first target domain of one target nucleic acid; 
 (2) a first primer sequence; and 
 (3) a 5′ ligation moiety; and 
 
 (ii) a second ligation probe comprising:
 (1) a second probe domain complementary to the second target domain of the target nucleic acid; 
 (2) a second primer sequence; and 
 (3) a 3′ ligation moiety; 
 
 (iii) a third ligation probe comprising:
 (1) a third probe domain complementary to the third target domain of the target nucleic acid; 
 (2) a third primer sequence; and 
 (3) a 5′ ligation moiety; 
 
 (iv) a fourth ligation probe comprising:
 (1) a fourth probe domain complementary to the fourth target domain of the target nucleic acid; 
 (2) a fourth primer sequence; and 
 (3) a 3′ ligation moiety; 
 
   (c) ligating said first and second ligation probe to form a first ligation product and said third and fourth ligation probe and the fifth ligation probe to form a second ligation product without the use of a ligase enzyme;   (d) amplifying the first and second ligation products; and   (e) detecting the presence of the first and second ligation products, thereby assessing the degradation of the target nucleic acid.   
     
     
         45 . The method of  claim 44 , wherein each of the 5′ ligation moieties comprises DABSYL and each of the 3′ ligation moiety comprises phosphorothioate. 
     
     
         46 . The method of  claim 44 , wherein the lysis buffer comprises gunadinium hydrochloride (GuHCl). 
     
     
         47 . The method of  claim 46 , wherein the lysis buffer comprises 3M GuHCl. 
     
     
         48 . The method of  claim 44 , wherein the target nucleic acid is DNA. 
     
     
         49 . The method of  claim 33 , wherein the target nucleic acid is RNA. 
     
     
         50 . The method of  claim 44 , wherein the capture moiety of the target capture probe comprises a member selected from the group consisting of a capture nucleic acid, a bead and a bind partner of a binding partner pair. 
     
     
         51 . The method of  claim 44 , wherein the target nucleic acid further comprises a fifth target domain adjacent to a sixth target domain, wherein the fifth and sixth target domains are upstream of the third and fourth target domains and are at a known distance for the target capture domain,
 the probe set further comprising:
 (v) a fifth ligation probe comprising:
 (1) a fifth probe domain complementary to the fifth target domain of the target nucleic acid; 
 (2) a fifth primer sequence; and 
 (3) a 5′ ligation moiety; 
 
 (iv) a sixth ligation probe comprising:
 (1) a sixth probe domain complementary to the sixth target domain of the target nucleic acid; 
 (2) a sixth primer sequence; and 
 (3) a 3′ ligation moiety, 
 
   wherein (c) further comprises ligating the fifth and sixth ligation probes to form a third ligation product,   wherein (d) amplifying further comprises amplifying the third ligation product; and   wherein (e) detecting further comprises detecting the third ligation product.