Ultra-Sensitive Platform for Nucleic acid detection using a novel method, Scanning Digital polymerase chain reaction (PCR)

Abstract

A method for analyzing a target nucleic acid includes diluting nucleic acid targets and filling pico to femto-liter sized wells such that they contain a single target nucleic acid and one or more amplification reagents, amplifying the target in the individual wells, distinguishing wells containing amplicon from the target and amplicon from a variant of the target generated by polymerase error by using two differently labeled-hybridization probes, one hybridizing to the target and one hybridizing to a specific variant of the target; and analyzing target amplicons.

What is claimed is: 
     
         1 . A method for analyzing a target nucleic acid, the method comprising the steps of:
 diluting nucleic acid targets and filling pico to femto-liter sized wells such that they contain a single target nucleic acid and one or more amplification reagents;   amplifying the target in the individual wells;   distinguishing wells containing amplicon from the target and amplicon from a variant of the target generated by polymerase error by using two differently labeled-hybridization probes, one hybridizing to the target and one hybridizing to a specific variant of the target; and   analyzing target amplicons.   
     
     
         2 . The method according to  claim 1 , wherein said amplifying step is a polymerase chain reaction and the one or more amplification reagents includes one or more primer pairs. 
     
     
         3 . The method according to  claim 1 , wherein said distinguishing step comprises scanning a laser across each well individually. 
     
     
         4 . The method according to  claim 1 , wherein said analyzing step comprises detecting said amplicons by hybridization to detectably-labeled probes. 
     
     
         5 . The method according to  claim 1 , wherein said analyzing step is conducted on amplicon from wells that were not distinguished in said distinguishing step. 
     
     
         6 . The method according to  claim 1 , wherein said analyzing step comprises:
 determining a number of wells that contain only wild-type target;   determining a number of wells that contain only a variant of the target.   
     
     
         7 . The method according to  claim 6 , wherein presence of wells containing only said variant is indicative of a disease. 
     
     
         8 . The method according to  claim 7 , wherein the disease is cancer. 
     
     
         9 . The method according to  claim 6 , wherein the variant is an allelic variant. 
     
     
         10 . The method according to  claim 9 , wherein the allelic variant is a single nucleotide polymorphism.