Absolute PCR Quantification

Abstract

The present application provides methods and devices for absolute quantification of polymerase chain reaction target nucleic acids. In particular, the methods and devices of the present application provide for splitting a nucleic acid sample to be analyzed into small, isolated volumes, conducting the method of polymerase chain reaction (PCR) on said volumes, detecting PCR amplification products, analyzing said detected PCR amplification products, performing absolute quantification of the PCR target and presenting said quantification results.

1 - 57 . (canceled) 
     
     
         58 . A method of sampling a target nucleic acid, comprising:
 a) providing a sample tray comprising a plurality of isolated reaction zones, wherein each isolated reaction zone defines a specific reaction sub-volume, wherein the reaction sub-volumes of said different isolated reaction zones are variable reaction sub-volumes;   b) exposing the sample tray to a sample comprising one or more target nucleic acid molecules under conditions wherein said sample fills said plurality of isolated reaction zones;   c) detecting the target nucleic acid in said isolated reaction zones.   
     
     
         59 . The method of  claim 58 , wherein said variable reaction sub-volumes are serial reaction sub-volumes. 
     
     
         60 . The method of  claim 59 , wherein the serial reaction sub-volumes differ between isolated reaction zones by factors of 2. 
     
     
         61 - 63 . (canceled) 
     
     
         64 . The method of  claim 59 , wherein the serial reaction sub-volumes differ between isolated reaction zones by factors of 1000. 
     
     
         65 . (canceled) 
     
     
         66 . The method of  claim 58 , wherein said detecting comprises performing a nucleic acid detection reaction. 
     
     
         67 . The method of  claim 66 , wherein said nucleic acid detection reaction comprises a nucleic acid amplification reaction. 
     
     
         68 . The method of  claim 67 , wherein said nucleic acid amplification reaction is a polymerase chain reaction. 
     
     
         69 . The method of  claim 68 , wherein said detecting is conducted after the completion of said polymerase chain reaction. 
     
     
         70 . The method of  claim 58 , wherein said detecting comprises detection of fluorescence. 
     
     
         71 . The method of  claim 70 , wherein said detecting comprises use of an oligonucleotide probe comprising a fluorescent moiety. 
     
     
         72 . The method of  claim 71 , wherein said oligonucleotide probe further comprises a quencher moiety. 
     
     
         73 . The method of  claim 70 , wherein said detecting comprises use of a nucleic acid intercalating dye. 
     
     
         74 . The method of  claim 68 , wherein said polymerase chain reaction is conducted for a predetermined number of cycles, and wherein said detecting is conducted after completion of said predetermined number of cycles.