Absolute PCR quantification
View Patent ↗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. 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:
M 0 =N sub-volumes ×[−ln( P )]
wherein M 0 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.
2. The method of claim 1 , wherein said multiple vessel array is a multiple capillary array, and wherein said reaction vessels are capillary reaction vessels.
3. The method of claim 1 , wherein said diluting comprises serial diluting.
4. The method of claim 3 , wherein at least 200 sub-volumes are made in step c).
5. The method of claim 3 , wherein sub-volumes in said multiple vessel array comprise between 1 and 10 nanoliters.
6. The method of claim 5 , wherein said reagents are dried prior to exposure to said sample.
7. The method of claim 1 , wherein said determining is conducted after the completion of a plurality of cycles of said polymerase chain reaction.
8. The method of claim 1 or claim 2 , wherein said detectable signal comprises fluorescence.
9. The method of claim 1 , wherein said reagents comprise an oligonucleotide probe comprising a fluorescent moiety.
10. The method of claim 9 , wherein said oligonucleotide probe further comprises a quencher moiety.
11. The method of claim 1 or claim 2 , wherein said reagents comprise a nucleic acid intercalating dye.
12. The method of claim 1 or 2 , wherein a standard deviation in the number of target molecules in a sample is calculated using the equation:
σ N =√{square root over ( N sub-volumes P (1 −P ))}
wherein σ N is the standard deviation.
13. The method of claim 1 or 2 , wherein said sample is a test sample, further comprising providing a negative control sample lacking said target nucleic acid, wherein said negative control sample is treated identically to said test sample, wherein the Poisson parameter for said test sample is estimated using the equation:
λ
EST
=
ln
[
P
CONTROL
(
0
)
/
P
SAMPLE
(
0
)
]
wherein:
λ EST is the estimated Poisson parameter for said test sample;
P CONTROL (0) is the percent of reaction vessels lacking signal in the control negative assay; and
P SAMPLE (0) is the percent of reaction vessels lacking signal in said test DNA sample.
14. The method of claim 2 , further comprising communicating the determined number of capillary reaction vessels containing reactions producing said detectable signal to a computer, wherein a calculation is performed by said computer.