Method and system for temperature correction in thermal melt analysis
AssigneeCanon U.S. Life Sciences, Inc.
InventorWeidong Cao
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Abstract
The present invention relates to methods and systems for temperature correction in thermal melt analysis. More specifically, embodiments of the invention relate to the correction of the melting temperature (Tm) observed for a sample nucleic acid due to the effect of the nucleic acid concentration.
Claims12
A computerized method for correcting a measured melting temperature of a nucleic acid in a sample, wherein the melting temperature refers to the dissociation of double stranded DNA into single stranded DNA, wherein a first reference sample comprising a nucleic acid, and a second nucleic acid containing sample are utilized, comprising:
- a) determining a ratio of the concentration of the nucleic acid in the first reference sample to the concentration of the nucleic acid in the second nucleic acid containing sample, wherein the first and second samples contain identical nucleic acids in different concentrations;
- b) using an imaging system and a temperature control system in communication with a computer to measure the melting temperature of the nucleic acid in the second nucleic acid containing sample (Tm second sample measured) and the melting temperature of the nucleic acid in the first reference sample (Tm1);
- c) determining a temperature compensation value from said ratio and determining the change of enthalpy (ΔH0) of the nucleic acid in the first reference sample,
- wherein the temperature compensation value is ΔTm, wherein ΔTm is Tm2−Tm1,
- wherein Tm1 is the measured melting temperature of the nucleic acid in the first reference sample and
- Tm2 is calculated by the equation
- 1
- T
- m
-
-
- 2
- =
- 1
- T
- m
-
-
- 1
- -
- R
-
-
- ln
-
- (
- C
-
-
- 1
- C
-
-
- 2
- )
- Δ
-
-
- H
- 0
- ,
- wherein R is the gas constant and C1/C2 is the ratio of the concentration of the nucleic acid in the first reference sample to the concentration of the nucleic acid in the second nucleic acid containing sample;
- wherein the determining a temperature compensation value is performed by the computer;
- and
- d) correcting the measured melting temperature of the nucleic acid in the second nucleic acid containing sample with the temperature compensation, wherein the correcting the measured melting temperature is performed by the computer based upon the formula Tm second sample corrected=Tm second sample measured−ΔTm.
A system for correcting a melting temperature of a nucleic acid in a sample, wherein the melting temperature refers to the dissociation of double stranded DNA into single stranded DNA, comprising:
- (a) a first reference sample and a second sample, the first and second samples containing identical nucleic acids in different concentrations;
- (b) an imaging system and a temperature control system in communication with the first reference sample and the second sample to measure data associated with the nucleic acid melting process;
- (c) a computer system in communication with the imaging system and the temperature control system, the computer system to determine the melting temperature of the nucleic acid in the first reference sample (Tm1) and a melting temperature of the nucleic acid in the second sample (Tm second sample measured) based upon the data associated with the nucleic acid melting process;
- wherein the computer system determines a temperature compensation value from a ratio of the concentration of the nucleic acid in a first reference sample to the concentration of the nucleic acid in the second nucleic acid containing sample and the determined melting temperature of the nucleic acid in the first reference sample and a change of enthalpy (ΔH0) of the nucleic acid in the reference sample,
- wherein the temperature compensation value is ΔTm, wherein ΔTm is Tm2−Tm1,
- wherein Tm1 is the measured melting temperature of the nucleic acid in the first reference sample and Tm2 is the melting temperature resulting from a difference in concentration of the nucleic acid,
- wherein Tm2 is calculated by the equation
- 1
- T
- m
-
-
- 2
- =
- 1
- T
- m
-
-
- 1
- -
- R
-
-
- ln
-
- (
- C
-
-
- 1
- C
-
-
- 2
- )
- Δ
-
-
- H
- 0
- ,
- wherein R is the gas constant and C1/C2 is the ratio of the concentration of the nucleic acid in the first reference sample to the concentration of the nucleic acid in the second nucleic acid containing sample;
- and
- wherein the computer system subtracts the temperature compensation value from the measured melting temperature of the nucleic acid in the second nucleic acid containing sample to provide a corrected melting temperature of the nucleic acid in the second nucleic acid containing sample based upon the formula Tm second sample corrected=Tm second sample measured−ΔTm.
Dependencies are inferred from the claim text.