Workflow for detection of ligands using nucleic acids

Abstract

This application relates to methods for ligating oligonucleotides having complementarity to a target nucleic acid, and amplifying the ligated oligonucleotides, where ligation and amplification occur in the same reaction mixture.

1 . A method for ligating at least two oligonucleotides to produce a ligated oligonucleotide and amplifying the ligated oligonucleotide, wherein ligation and amplification occur in a single reaction mixture. 
     
     
         2 . The method of  claim 1  whereby a ligand is detected in a test sample, the method comprising the steps of, in combination:
 a) contacting a target protein or analyte with at least a first and second probe, each probe having binding specificity for the protein or analyte, and being adjoined to at least one type of oligonucleotide; 
 b) ligating the oligonucleotides on the first and second probes to one another using a ligase to produce a target nucleic acid and amplifying the target nucleic acid; and, 
 c) detecting the amplified target nucleic acid. 
 
     
     
         3 . The method of  claim 1  or  2 , wherein a portion of at least one of said probes is an antibody. 
     
     
         4 . The method of  claim 3 , wherein a portion of each of said first and second probes are antibodies. 
     
     
         5 . The method of any one of  claims 1 - 4 , wherein at least one of said oligonucleotides comprises at least three nucleotides. 
     
     
         6 . The method of any one of  claims 1 - 5 , wherein at least one of said oligonucleotides consists of three nucleotides. 
     
     
         7 . The method of any one of  claims 1 - 6 , wherein said oligonucleotides are ligated using a small footprint ligase (SFL). 
     
     
         8 . The method of  claim 7 , wherein said small footprint ligase is contacted with adenosine triphosphate prior to use. 
     
     
         9 . The method of any one of  claims 1 - 8 , wherein said ligated oligonucleotide is amplified using the polymerase chain reaction (PCR). 
     
     
         10 . The method  claim 9 , wherein said amplified ligated oligonucleotide is detected using quantitative PCR (qPCR). 
     
     
         11 . The method of any one of  claims 1 - 10 , wherein a ligase is used for ligation, and said ligase is inactivated prior to amplification. 
     
     
         12 . The method of  claim 11 , wherein said ligase is inactivated by heat. 
     
     
         13 . The method of  claim 11  or  12 , wherein said ligase is selected from the group consisting of a nucleic acid ligase, oligonucleotide ligase, DNA ligase, T3 DNA ligase, T4 DNA ligase, T5 DNA ligase, T7 DNA ligase, vaccinia virus DNA ligase,  E. coli  DNA ligase, mammalian DNA ligase I, mammalian DNA ligase II, mammalian DNA ligase III, Tth DNA ligase, KOD DNA ligase, a thermostable DNA ligase, RNA ligase, T4 RNA ligase, bacteriophage RB69 RNA ligase,  Autographa californica  nuclearpolyhedrosis virus RNA ligase, a thermophilic RNA ligase, bacteriophage RM378 RNA ligase, bacteriophage TS2126 RNA ligase, a small-footprint DNA ligase (SFL), the ligase of SEQ ID NO.: 77, the ligase of SEQ ID NO.: 78, the ligase of SEQ ID NO.: 79, the ligase of SEQ ID NO.: 80, the ligase of SEQ ID NO.: 81, the ligase of SEQ ID NO.: 82, a derivative thereof, a fragment thereof, and combinations thereof. 
     
     
         14 . A method for detecting a target in a sample wherein:
 a) binding a first and a second probe, each of which binds specifically to the target, wherein each of the probes comprises an oligonucleotide portion or tail;   b) ligating the first and second oligonucleotide tails thereby producing a ligated oligonucleotide template; and,   c) performing a polymerase chain reaction (PCR) of the oligonucleotide template across the first and second oligonucleotide to quantify the said template.   
     
     
         15 . The method of  claim 14 , wherein steps b and c are performed in the same reaction mixture. 
     
     
         16 . The method of  claim 14  or  15 , wherein a splint oligonucleotide is used to ligate said first and second oligonucleotide tails. 
     
     
         17 . The method of  claim 16 , wherein the 3′ and 5′ ends (which overlap with said first and second oligonucleotide tails, respectively) of said splint oligonucleotide are symmetrical or asymmetrical to one another. 
     
     
         18 . The method of  claim 16  or  17 , wherein said splint oligonucleotide is blocked at it's 3′-end. 
     
     
         19 . The method of any of  claims 16 - 18 , wherein said splint oligonucleotide is at least 6 nucleotides in length. 
     
     
         20 . The method of any of  claims 16 - 19 , wherein said 3′-end of said splint oligonucleotide overlaps with at least 3 nucleotides of said first oligonucleotide tail and/or said 5′-end of said splint oligonucleotide overlaps with at least 3 nucleotides of said second oligonucleotide tail. 
     
     
         21 . The method of any of  claims 14 - 20 , wherein the length of said first and second oligonucleotide tails is at least 3 nucleotides in length. 
     
     
         22 . The method of any of  claims 14 - 21 , wherein a small footprint ligase (SFL) is used to ligate said first and second oligonucleotide tails. 
     
     
         23 . The method of any of  claims 14 - 22 , wherein said first and/or second probes further comprises an antibody portion specific to said target. 
     
     
         24 . A method for detecting a target in sample comprising:
 a) binding a first and a second probe, wherein each probe binds specifically to the target, each of said probes comprise an oligonucleotide portion or tail;   b) ligating the oligonucleotides to produce a ligated oligonucleotide template and amplifying the template by PCR in a single step to produce an amplified template; and,   c) quantitating the amplified template   
     
     
         24 . The method of  claim 24 , wherein the probe further comprises an antibody. 
     
     
         25 . The method of  claim 24  or  25 , wherein said ligase is selected from the group consisting of a nucleic acid ligase, oligonucleotide ligase, DNA ligase, T3 DNA ligase, T4 DNA ligase, T5 DNA ligase, T7 DNA ligase, vaccinia virus DNA ligase,  E. coli  DNA ligase, mammalian DNA ligase I, mammalian DNA ligase II, mammalian DNA ligase III, Tth DNA ligase, KOD DNA ligase, a thermostable DNA ligase, RNA ligase, T4 RNA ligase, bacteriophage RB69 RNA ligase,  Autographa californica  nuclearpolyhedrosis virus RNA ligase, a thermophilic RNA ligase, bacteriophage RM378 RNA ligase, bacteriophage TS2126 RNA ligase, a small-footprint DNA ligase (SFL), the ligase of SEQ ID NO.: 77, the ligase of SEQ ID NO.: 78, the ligase of SEQ ID NO.: 79, the ligase of SEQ ID NO.: 80, the ligase of SEQ ID NO.: 81, the ligase of SEQ ID NO.: 82, a derivative thereof, a fragment thereof, and combinations thereof. 
     
     
         26 . The method of  claim 26 , wherein said ligase is a T4 DNA ligase. 
     
     
         27 . The method of  claim 26 , wherein the ligase is a small-footprint DNA ligase (SFL). 
     
     
         28 . The method of any one of  claims 24 - 27 , wherein said oligonucleotides are ligated using a splint oligonucleotide. 
     
     
         29 . The method of  claim 28 , wherein said splint oligonucleotide comprises a 3′ end of four to nine bases in length and a 5′ end of four to nine bases in length. 
     
     
         30 . The method of  claim 28  or  29 , wherein said 3′ and 5′ ends (which overlap with said first and second tails, respectively) of said splint oligonucleotide are symmetrical or asymmetrical to one another. 
     
     
         31 . The method of  claim 28 - 30 , wherein said splint oligonucleotide is blocked at it's 3′-end. 
     
     
         32 . The method of any one of  claims 23 - 31 , wherein the ligase is pre-enriched using ATP. 
     
     
         33 . The method of any one of  claims 23 - 32 , wherein said ligase is inactivated after ligation using a protease or heat. 
     
     
         34 . The method of any one of  claims 23 - 33 , wherein said template is quantified using real-time PCR. 
     
     
         35 . The method of  claim 34 , wherein said real-time PCR assay is a TaqMan assay. 
     
     
         36 . The method of  claim 1 ,  14 , or  24 , wherein said method provides at least about a one to three-fold dCt improvement over a typical PLA method or process. 
     
     
         37 . The method of  claim 1 ,  14 , or  24 , wherein said method provides about a two- to ten-fold improvement/increase in sensitivity over the typical process. 
     
     
         38 . The method of  claim 36  or  37 , wherein said typical method or process includes the use of a protease and/or dilution of the reaction mixture prior to PCR and said improved method of  claim 1 ,  14 , or  24  does not. 
     
     
         39 . The method of  claim 2 , wherein said oligonucleotides on said first and second probes, are at least partially complementary to one another