METHOD, PRODUCT, AND SYSTEM FOR QUANTIFYING THE METHYLATION STATUS OF A NUCLEIC ACID IN A SAMPLE

摘要

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.

主权项

1. 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.