Absolute PCR quantification

摘要

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.

主权项

1. A method of determining the number of target nucleic acid molecules in a sample, comprising:
a) providing a sample containing a plurality of target nucleic acid molecules, the total number of target nucleic acid molecules being unknown; b) diluting said sample to create a plurality of diluted samples; c) applying said diluted samples to a multiple vessel array comprising a plurality of reaction vessels, wherein each of said reaction vessels receives an essentially equal sub-volume of one of said diluted samples, wherein the combined volumes of said sub-volumes defines a tested volume, and wherein, as the result of said diluting, the number of said plurality of reaction vessels exceeds the number of target nucleic acid molecules in said tested volume; d) treating said multiple vessel array with polymerase chain reaction (PCR) amplification reagents and conditions to produce a detectable signal in the presence of said target nucleic acid; e) determining how many of said reaction vessels contain reactions that produce and do not produce said detectable signal; and f) determining the number of target nucleic acid molecules in said sample of step a) using the equation:
M0=Nsub-volumes×[−ln(P)] wherein M0 is the number of target molecules in said tested volume, wherein N is the number of sub-volumes, and wherein P is the percent of sub-volumes lacking said detectable signal.