Chemical ligation dependent probe amplification (clpa)

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 oligonucleotide 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 ligated oligonucleotide product. In one aspect, the ligation product is of variable length that correlates with a particular target. Following chemical ligation, the probes may be amplified and detected by capillary electrophoresis or microarray analysis.

1 . A method for detecting in a sample, comprising a plurality of sample nucleic acids of different sequence, the presence of at least one specific target nucleic acid sequence comprising a first and a second target domain, the domains located essentially adjacent to one another, comprising the steps of:
 a) contacting the sample nucleic acids with a plurality of different probes sets, each probe set comprising
 i. a first ligation probe comprising:
 1) a first probe domain substantially complementary to said first target domain; and 
 2) a first non-complementary region being essentially non-complementary to the said target nucleic acid 
 3) a 5′-ligation moiety; and 
 
 ii. second ligation probe comprising:
 1) a second probe domain substantially complementary to said second target domain; 
 2) a second non-complementary region, being essentially non-complementary to the said target nucleic acid 
 3) a 3′ ligation moiety; 
 
   wherein at least one of said ligation probe comprises a variable spacer sequence; and   b) ligating said first and second ligation probes in the absence of a ligase enzyme to form a ligation product;   c) amplifying said ligation product; and   d) detecting the presence of said ligation product.   
     
     
         2 . A method of  claim 1  wherein said target sequence is RNA and/or DNA. 
     
     
         3 . A method of  claim 1  wherein said target sequence comprises unpurified RNA 
     
     
         4 . The method of  claim 1  wherein said sample is derived from a mammalian body selected from the group consisting of blood, urine, saliva and feces. 
     
     
         5 .- 7 . (canceled) 
     
     
         8 . A method as in  claim 1  wherein said step of detection is by mass spectrometry. 
     
     
         9 . The method of  claim 1  wherein said 5′ ligation moiety on said first ligation probe is DAB SYL moiety and said 3′ ligation moiety on said second ligation probe is 3-phophorothioate moiety. 
     
     
         10 . A method as in  claim 1  wherein said first and second ligation probes each further comprising a universal primer sequence for amplification of said ligation product. 
     
     
         11 . The method of  claim 10  wherein one of the universal primers that binds said primer sequence contains a detectable label. 
     
     
         12 . (canceled) 
     
     
         13 . (canceled) 
     
     
         14 . A method for detecting in a sample, comprising a plurality of sample nucleic acids of different sequence, the presence of at least one specific target nucleic acid sequence comprising a first and a second target domain, the domains located essentially adjacent to one another, comprising the steps of:
 a) Contacting the sample nucleic acids with a plurality of different probes sets, each probe set comprising
 i) a first ligation probe comprising:
 1) a first probe domain substantially complementary to said first target domain; and 
 2) a first non-complementary region being essentially non-complementary to the said target nucleic acid 
 3) a 5′-ligation moiety; and 
 
 ii) second ligation probe comprising:
 1) a second probe domain substantially complementary to said second target domain; 
 2) a second non-complementary region, being essentially non-complementary to the said target nucleic acid; and 
 3) a 3′ ligation moiety; 
 
   wherein at least one of said first and second ligation probes further comprises an anchor sequence   b) capturing said ligation product on a microarray substrate comprising a capture probe substantially complementary to said anchor sequence; and   c) detecting the presence of said ligated product.   
     
     
         15 . A method as in  claim 14  wherein said first and second ligation probes each further comprising a universal primer sequence for amplification of said ligation product wherein one of the universal primers that binds said primer sequence contains a detectable label wherein said detectable label is selected from the group consisting of a fluorescent label, an electrochemical label and a magnetic label. 
     
     
         16 . A method for detecting in a sample, comprising a plurality of sample nucleic acids of different sequence, the presence of at least one specific target nucleic acid sequence comprising a first and a second target domain, and a third domain located between the first and second domains, the domains located essentially adjacent to one another, comprising the steps of:
 b) contacting the sample nucleic acids with a plurality of different probes sets, each probe set comprising
 i. a first ligation probe comprising:
 1) a first probe domain substantially complementary to said first target domain; and 
 2) a first non-complementary region being essentially non-complementary to the said target nucleic acid 
 3) a 5′-ligation moiety; and 
 
 ii. second ligation probe comprising:
 1) a second probe domain substantially complementary to said second target domain; 
 2) a second non-complementary region, being essentially non-complementary to the said target nucleic acid 
 3) a 3′ ligation moiety; 
 
 iii. a third ligation probe comprising
 1) a third probe domain substantially complementary to the said third target domain 
 2) a 3′ and a 5′ ligation moiety; 
 
   b) ligating said first ligation probe, said second ligation probe, and said third ligation probe in the absence of a ligase enzyme to form a ligation product;   c) amplifying said ligation product; and   c) detecting the presence of said ligated product.   
     
     
         17 . A method of  claim 16  wherein said target sequence is RNA and/or DNA. 
     
     
         18 . The method of  claim 16  wherein said sample is derived from a mammalian body selected from the group consisting of blood, urine, saliva and feces. 
     
     
         19 . 22 . (canceled) 
     
     
         23 . A method as in  claim 16  wherein said first and second ligation probes each further comprising a universal primer sequence for amplification of said ligation product. 
     
     
         24 . The method of  claim 16  wherein one of the universal primers that binds said primer sequence contains a detectable label. 
     
     
         25 .- 31 . (canceled)