Methods of detecting polynucleotide kinase and its use as a label

Abstract

Methods of detecting or measuring the activity of polynucleotide kinase are disclosed as well as methods of detecting an analyte in an assay using polynucleotide kinase as a label on a member of a specific binding pair. the methods rely on the phosphorylation of an oligonucleotide followed by ligation of the oligonucleotide 5′-phosphate onto another template-bound oligonucleotide. The presence of the ligated product signals the presence of polynucleotide kinase. In preferred embodiments, phosphorylation of an oligonucleotide enables the consecutive ligation of a set of oligonucleotides. The oligonucleotides so ligated can be detectably labeled with, for example, other enzymes to provide highly sensitive detection methods.

1 . A method comprising: 
 a)) reacting polynucleotide kinase, a nucleotide triphosphate and an oligonucleotide having a 5′-OH group and comprising a sequence S2 to phosphorylate oligonucleotide S2 at the 5′-OH group;    b) providing a reaction mixture comprising 
 i) oligonucleotide 5′-phosphate S2,  
 ii) a single stranded nucleic acid template comprising contiguous sequence regions C1-Cn, wherein n is an integer greater than 2,  
 iii) a first oligonucleotide having a sequence S1 which is hybridized to region C1 of the template,  
 iv) a plurality of oligonucleotide 5′-phosphates having sequences S3-Sn, wherein each oligonucleotide 5′ phosphate is complementary to one region of the template C3-Cn, and  
 v) a ligase and a cofactor for the ligase;  
   c) ligating with the ligase oligonucleotide S1 and oligonucleotide 5′-phosphate S2 and ligating at least one of the plurality of oligonucleotide 5′-phosphates S3-Sn in a contiguous manner onto oligonucleotide S2 to form a ligation product in one continuous process under conditions which do not permit stable hybridization of the oligonucleotides S2-Sn, wherein oligonucleotide S1 contains at least about five bases more than each of oligonucleotides S2-Sn, and wherein ligation of oligonucleotide 5′-monophosphates S2-Sn only occurs if oligonucleotide S2 is phosphorylated by polynucleotide kinase.    
   
   
       2 . The method of  claim 1  wherein each of the oligonucleotide 5′-phosphates consists of the same number of bases and the number of bases is from 2 to 20.  
   
   
       3 . The method of  claim 1  wherein wherein each of the oligonucleotide 5′-phosphates consists of the same number of bases and the number of bases is from 4 to 12.  
   
   
       4 . The method of  claim 1  wherein the nucleotide triphosphate used is γ32P-ATP.  
   
   
       5 . The method of  claim 1  wherein at least some of the oligonucleotide 5′-phosphates comprise a detectable label.  
   
   
       6 . The method of  claim 5  wherein the detectable label is selected from radioisotopes, chemiluminescent labels, fluorescent labels, colorimetric labels, enzymes, binding proteins, antigens, antibodies and haptens.  
   
   
       7 . The method of  claim 1  wherein the ligated product is unlabeled and is detected by a technique which determines its length or number of bases.  
   
   
       8 . The method of  claim 7  wherein the label is an enzyme and comprising the further steps of reacting the enzyme label present in the ligated product with a substrate for the enzyme and detecting the product of the reaction between the enzyme and the substrate.  
   
   
       9 . The method of  claim 8  wherein the label enzyme is selected from alkaline phosphatase, β galactosidase, β-glucuronidase, glucose oxidase and horseradish peroxidase.  
   
   
       10 . The method of  claim 8  wherein the substrate is selected from substrates which produce a colored product, a fluorescent product, chemiluminescence or bioluminescence.  
   
   
       11 . The method of  claim 1  wherein one of the oligonucleotides selected from S4-Sn is a nonextendable oligonucleotide having a 3′-terminus which can not take part in a ligation reaction.  
   
   
       12 . The method of  claim 1  wherein the nonextendable oligonucleotide is selected from oligonucleotides which have a dideoxy base at the 3′-end and oligonucleotides which have a blocked 3′-OH group at the 3′-end.  
   
   
       13 . (canceled)  
   
   
       14 . The method of  claim 1  wherein the template is immobilized onto a solid support.  
   
   
       15 . The method of  claim 1  wherein the solid support comprises magnetic particles.  
   
   
       16 . The method of  claim 15  wherein the solid support containing the template and hybridized ligated oligonucleotide product is separated from any unhybridized oligonucleotides.  
   
   
       17 . The method of  claim 1  wherein the polynucleotide kinase is T4 polynucleotide kinase.  
   
   
       18 . The method of  claim 1  wherein the ligase enzyme is selected from T4 ligase, T7 ligase, Tth ligase, Taq ligase and  E. coli  DNA ligase.  
   
   
       19 . The method of  claim 18  wherein the ligase enzyme is T4 DNA ligase.  
   
   
       20 . The method of  claim 1  used for detecting or measuring the activity of polynucleotide kinase.  
   
   
       21 . The method of  claim 1  wherein the single stranded nucleic acid template is produced by separating a double stranded nucleic acid.  
   
   
       22 - 51 . (canceled)  
   
   
       52 . The method of  claim 1  wherein steps a-c are performed in a single reaction mixture in one process.  
   
   
       53 . A method comprising: 
 a) providing a reaction mixture comprising polynucleotide kinase, a nucleotide triphosphate and a plurality of oligonucleotides S2-Sn, each oligonucleotide having a 5′-OH group, a single stranded nucleic acid template comprising contiguous sequence regions C1-Cn, wherein n is an integer greater than 2, a first oligonucleotide having a sequence S1 which is hybridized to region C1 of the template, a ligase, and a cofactor for the ligase;    b) in one process, phosphorylating oligonucleotides S2-Sn to form oligonucleotide 5′-phosphates S2-Sn and ligating, with the ligase, oligonucleotide S1 and oligonucleotide 5′-phosphate S2, and ligating at least one of the plurality of oligonucleotide 5′-phosphates S3-Sn in a contiguous manner onto oligonucleotide S2 to, form a ligation product in one continuous process under conditions which do not permit stable hybridization of the oligonucleotides S2-Sn, wherein oligonucleotide S1 contains at least about five bases more than each of oligonucleotides S2-Sn, and wherein ligation of oligonucleotide 5′-monophosphates S2-Sn only occurs if oligonucleotide S2 is phosphorylated by polynucleotide kinase.    
   
   
       54 . The method of  claim 53  wherein each of the plurality of oligonucleotide 5′-phosphates S3-Sn is ligated onto oligonucleotide S2.