US 2014/0273254 A1Published application

Method, product, and system for quantifying the methylation status of a nucleic acid in a sample

AssigneeThermo Fisher Scientific Inc.

InventorJaakko Kurkela

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Abstract

Methods and systems are described for quantifying the methylation status of nucleic acids in a sample utilizing standardized curves derived from methylation-sensitive HRM data. The standardized curves are generated from HRM curves from a plurality of samples, each sample having a known but different methylation status. In one embodiment, the first negative derivative of each HRM curve from the known samples is plotted and a first value corresponding with a first melt peak and a second value corresponding with a second melt peak from the negative derivative plots are identified. The slope of a line connecting the first and second values for each sample is calculated and used to identify a slope data point that is plotted to generate the standardized curve. In another embodiment, a threshold line that intersects the plurality of HRM curves is generated and the standardized curve is generated from the intersection data points.

Claims25

Dependent claims

A method of analyzing HRM data to quantify the methylation status of nucleic acids in a sample comprising:

obtaining a first curve from HRM data from a first sample having a first known methylation status of nucleic acids,
plotting the first negative derivative of the first curve,
identifying a first value corresponding with a first melt peak and a second value corresponding with a second melt peak from the first curve,
calculating a first slope of a line connecting the first value with the second value from the first curve, and
identifying a first slope data point corresponding to the first slope and the first known methylation status of nucleic acids in the first sample;
obtaining a second curve from HRM data from a second sample having a second known methylation status of nucleic acids,
plotting the first negative derivative of the second curve,
identifying a first value corresponding with a first melt peak and a second value corresponding with a second melt peak from the second curve,
calculating a second slope of a line connecting the first value with the second value from the second curve, and
identifying a second slope data point corresponding to the second slope and the second known methylation status of nucleic acids in the second sample;
and
generating a standard curve with the first slope data point and the second slope data point.

9 more elements

A method of analyzing HRM data to quantify the methylation status of nucleic acids in a sample, the method comprising:

obtaining a first curve with HRM data collected from a first sample having a first known methylation status of nucleic acids;
obtaining a second curve with HRM data collected from a second sample having a second known methylation status of nucleic acids that is greater than the first known methylation status of nucleic acids;
obtaining a third curve with HRM data collected from a third sample having a third known methylation status of nucleic acids that is greater than the second known methylation status of nucleic acids;
generating a threshold line that intersects the first curve at a first intersection data point, the second curve at a second intersection data point, and the third curve at a third intersection data point;
and
plotting a standard curve with the first data point, the second data point, and the third data point.

3 more elements

Dependencies are inferred from the claim text.