Determination of single peak melting temperature by PCR analogy and double sigmoid equation
AssigneeRoche Molecular Systems, Inc.
InventorsRonald T. Kurnik, Thomas Thurnherr
Life of this patent
Estimating the term…
Abstract
Systems and methods for determining melting temperatures, Tm, for DNA from melt curve data. The systems and methods also allow for quantitative determination of gene amount based on peak height. A PCR analogy is used to perform quantization of an acquired melting curve dataset. The melting curve is transformed using a horizontal flip and a horizontal translation, and a double sigmoid equation is then fit to the data. Inverse translation and inverse horizontal flip transforms are applied to the equation to produce an equation based solution of the melt curve dataset. The equation based solution of the melt curve is then used to determine the first derivative (e.g., Tm value) and peak height.
Claims32
A computer-implemented method of determining a melting temperature, Tm, of DNA, the method comprising:
- receiving a dataset representing a melt curve for a DNA sample, the dataset including a plurality of data points represented as a sequence of data values {(X1,Y1), (X2,Y2) . . . (Xn−1,Yn−1), (Xn,Yn)}, where X represents a temperature (T) value and Y represents a fluorescence intensity value;
- determining, with a computer system comprising a processor, an analytical expression that fits the dataset, wherein the analytical expression is determined by:
- calculating an approximation of a curve that fits the dataset by applying a regression process to a double sigmoid function to determine parameters of the double sigmoid function, the double sigmoid function having the form:
- a
- +
- bx
- +
- c
- (
- 1
- +
- exp
- -
- d
-
- (
- x
- -
- e
- )
- )
-
- (
- 1
- +
- exp
- -
- f
-
- (
- x
- -
- g
- )
- )
- ,
- where a, b, c, d, e, f, and g are the parameters;
- determining a derivative curve by taking the derivative of the analytical expression with respect to X;
- determining a value Xmax corresponding to a maximum derivative (dY/dX) value of the derivative curve;
- and
- outputting the value Xmax , wherein the value Xmax represents a melting temperature, Tm, of the DNA sample.
A non-transitory computer readable medium including code for controlling a processor to determine a melting temperature, Tm, of DNA, the code including instructions to:
- receive a dataset representing a melt curve for a DNA sample, the dataset including a plurality of data points represented as a sequence of data values {(X1,Y1), (X2,Y2) . . . (Xn−1,Yn−1), (Xn,Yn)}, where X represents a temperature (T) value and Y represents a fluorescence intensity value;
- determine an analytical expression that fits the dataset, wherein the analytical expression is determined by:
- calculating an approximation of a curve that fits the dataset by applying a regression process to a double sigmoid function to determine parameters of the double sigmoid function, the double sigmoid function having the form:
- a
- +
- bx
- +
- c
- (
- 1
- +
- exp
- -
- d
-
- (
- x
- -
- e
- )
- )
-
- (
- 1
- +
- exp
- -
- f
-
- (
- x
- -
- g
- )
- )
- ,
- where a, b, c, d, e, f, and g are the parameters;
- determine a derivative curve by taking the derivative of the analytical expression with respect to X;
- determine a value Xmax corresponding to a maximum dY/dX value of the derivative curve;
- and
- output the value Xmax , wherein the value Xmax represents a melting temperature, Tm, of the DNA sample.
A kinetic Polymerase Chain Reaction (PCR) system, comprising:
- a kinetic PCR analysis module that generates a melt curve dataset representing a DNA melt curve, said dataset including a plurality of data points represented as a sequence of data values {(X1,Y1), (X2,Y2) . . . (Xn−1,Yn−1), (Xn,Yn)}, where X represents a temperature (T) value and Y represents a fluorescence intensity value;
- and
- a processor adapted to process the melt curve dataset to determine a Tm value, by:
- determining, with a computer system comprising a processor, an analytical expression that fits the dataset, wherein the analytical expression is determined by:
- calculating an approximation of a curve that fits the dataset by applying a regression process to a double sigmoid function to determine parameters of the double sigmoid function, the double sigmoid function having the form:
- a
- +
- bx
- +
- c
- (
- 1
- +
- exp
- -
- d
-
- (
- x
- -
- e
- )
- )
-
- (
- 1
- +
- exp
- -
- f
-
- (
- x
- -
- g
- )
- )
- ,
- where a, b, c, d, e, f, and g are the parameters;
- determining a derivative curve by taking the derivative of the analytical expression with respect to X;
- determining a value Xmax corresponding to a maximum derivative (dY/dX) value of the derivative curve;
- and
- outputting the value Xmax, wherein the value Xmax represents a melting temperature, Tm, of the DNA sample.
Dependencies are inferred from the claim text.