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Determination of single peak melting temperature by PCR analogy and double sigmoid equation

A technology of melting temperature and melting curve, applied in the field of data processing, can solve problems such as no benefit of treatment

Active Publication Date: 2010-11-10
F HOFFMANN LA ROCHE & CO AG
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Patients whose KRAS gene is wild-type will benefit from the treatment, however, if the patient has a mutation in this gene, the treatment will not benefit

Method used

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  • Determination of single peak melting temperature by PCR analogy and double sigmoid equation
  • Determination of single peak melting temperature by PCR analogy and double sigmoid equation
  • Determination of single peak melting temperature by PCR analogy and double sigmoid equation

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Embodiment Construction

[0032] The present invention provides systems and methods for determining the melting temperature, Tm, of DNA from melting curve data. The described systems and methods also allow quantitative determination of gene numbers based on peak heights.

[0033] exist figure 1 An example of a melting curve in the context of the PCR method is shown in . figure 1 The curve shown in is a typical melting curve plot, which was generated after PCR experiments in the case where only one genotype was present. In this curve, the fluorescence intensity decreases as a function of increasing temperature.

[0034] Such as figure 1 As shown, data from a typical melting curve can be represented in a two-dimensional coordinate system, eg, temperature defines the x-axis and accumulated polynucleotide indicator defines the y-axis. Typically, the indicator of accumulated polynucleotides is a fluorescence intensity value, since the use of fluorescent labels is perhaps the most widely used labeling sc...

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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.

Description

Background of the invention [0001] The present invention relates generally to the processing of data representative of melting characteristics of oligonucleotides, and more particularly to systems and methods for determining the melting temperature of an oligonucleotide sample based on melting curve data. [0002] Determination of melting temperature is an important method for distinguishing genotypes. In a typical single-step, homogeneous, closed-tube method for determining the melting temperature of DNA or oligonucleotides, double-strand-specific DNA dyes (e.g., SYBRGreen I, Molecular Probes, Eugene, Oregon) or labeled oligonucleotides Used in real-time PCR to monitor product formation (Wittwer CT, et al., BioTechniques 1997; 22: 130-8) and melting temperature (Ririe KM, et al., Anal. Biochem 1997; 245: 154-60) . Typically, amplification is performed using a thermostable enzyme that is stable to DNA denaturing temperatures and at least one pair of oligonucleotide primers. ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G06F19/00G06F17/17C12Q1/68G16B40/10G16B20/20
CPCC12Q1/6816G06F19/18G06F19/24G16B20/00G16B40/00G16B40/10G16B20/20C12Q2527/107C12Q2537/165
Inventor R·T·库尼克T·特恩赫尔
Owner F HOFFMANN LA ROCHE & CO AG