System and method for melting curve normalization

Patent metadata

Field	Value
Publication number	US-2017372002-A1
Application number	US-201715631832-A
Country	US
Kind code	A1
Filing date	Jun 23, 2017
Priority date	Jun 23, 2016
Publication date	Dec 28, 2017
Grant date	—

Abstract

The present invention relates to methods for the analysis of nucleic acids present in biological samples, and more specifically to normalize a high resolution melt curve to assist in the identification of one or more properties of the nucleic acids. The present invention provides methods and systems that incorporate a background identification algorithm according to invention principles using raw melt curve data to identify reactions that are unrelated actual DNA melt reactions. Furthermore, a web-based application for analyzing experimental data is provided. The raw experimental data obtained from a variety of instruments is processed and analyzed on a server and presented to a user through a user interface (UI).

First claim

1 . A method for removing a background signal from a DNA melting curve on a device having at least one DNA sample and which includes a thermal system in communication with the device that continuously increases a temperature of the at least one DNA sample to cause a DNA melting reaction resulting in denaturing dsDNA to ssDNA, the method comprising:
(a) generating a melting curve F(T) for a DNA sample, wherein F is a fluorescence signal indicative of a DNA denaturation process and T is the temperature of the DNA sample; (b) providing a mathematical model to fit to the melting curve F(T), the mathematical model represented by a sum of a background reaction term and at least one DNA melting reaction term, each DNA melting reaction term being indicative of the DNA denaturation process and the background reaction term being indicative of the background signal; (c) fitting the mathematical model to the melting curve by calculating model parameters for a model curve; (d) estimating a maximum difference between the melting curve F(T) and the model curve and a temperature corresponding to the maximum difference; (e) iteratively adding one DNA melting reaction term at a time to the sum representing the mathematical model and refitting the sum to the melting curve F(T) to recalculate all model parameters in response to the difference between the melting curve and the model curve estimated in step (d) being greater than a threshold, wherein an initial temperature parameter for each newly added DNA melting reaction term equals to the temperature corresponding to the maximum difference between the melting curve and the model curve obtained in step (d); (f) stopping the iteration process of step (e) when the difference between the melting curve and the model curve is less than the threshold; and (g) subtracting the background reaction term having parameters estimated in the last iteration of step (e) from the melting curve F(T).

2 . The method of claim 1 , wherein the mathematical model to fit to the melting curve F(T) is represented by

F
total



(

T
;
Θ

)

=

∑

i
=
1

M



α
i



F
i



(

T
;

Θ
i

)




such



that

∑

i
=
1

M



α
i

=

1



and




α
i

≥

0



for



all



i

where T is temperature, F total (T; Θ) is a total fluorescence, F i (T;Θ i ) is a fluorescence of the i th reaction model, Θ i is a set of parameters for the i th reaction model, α i is a coefficient indicative of a contribution of the i th reaction model to the total fluorescence F total (T; Θ), and Θ is a collection of all parameters {α i , Θ i :iε1, . . . , M}.

3 . The method of claim 2 , wherein F i (T i ;Θ i ) is the fluorescence of the i th DNA melting reaction and is represented by

F


(
T
)

=

4
+

h


(
T
)

-

h
2



(
T
)

+

8


h


(
T
)

4

and the background reaction is represented by

F


(
T
)

=

1

1
+

h


(
T
)

where

h


(
T
)

=

exp


Assignees

• Canon Us Life Sciences Inc

Inventors

• Yang Yang
• Paquerault Sophie
• Denney Bradley Scott
• Charlton Lance
• Paek Jeanette
• Seikh Attaullah
• Parthasarathy Vyshnnavi
• Pearson Ken
• Huang Hung
• Uppal Tejinder

Classifications

• G16B45/00

  ICT specially adapted for bioinformatics-related data visualisation, e.g. displaying of maps or networks · CPC title

• B01L2300/023

  Sending and receiving of information, e.g. using Bluetooth® · CPC title

• B01L3/5027

  by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip · CPC title

• G01N2035/00881

  network configurations · CPC title

• G01N2035/00366

  Several different temperatures used · CPC title