US 10,591,364 B2Granted

Thermal calibration

AssigneeCanon U.S.A., Inc.

InventorsFernando DeCastro, Renee Howell, Sami Kanderian, Johnathan S. Coursey, Kenton C. Hasson, Scott Sundberg

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Abstract

The present invention relates to the use of one or more amplicons as temperature calibrators. In some embodiments, the calibrators may be used to calibrate the temperature of a microfluidic channel in which amplification and/or melt analysis is performed. In some embodiments, the amplicons may be genomic, ultra conserved elements and/or synthetic. The amplicon(s) may have a known or expected melt temperature(s). The calibrators may be added to primers of study or may follow or lead the primers of study in the channel. The amplicon(s) may be amplified and melted, and the temperature(s) at which the amplicon(s) melted may be determined. The measured temperature(s) may be compared to the known temperature(s) at which the amplicon(s) was expected to melt. The difference(s) between the measured and expected temperatures may be used to calibrate/adjust one or more temperature control elements used to control and/or detect the temperature of the channel.

Claims17

Dependent claims

A method of calibrating a temperature sensor measuring the temperature of a thermal melt in a microfluidic channel present in a microfluidic system, the method comprising:

(a) introducing into the microfluidic channel a sample consisting essentially of human genomic DNA, and reagents necessary for amplifying the human genomic DNA, comprising a polymerase, dNTPs, a fluorescent dye, at least a pair of primers for one or more nucleic acid sequences of interest found within the human genomic DNA and a pair of primers for a calibrant amplified from the human genomic DNA, wherein the primers for the calibrant are (i) SEQ ID Nos:
7 and 8 or (ii) SEQ ID Nos:
9 and 10;
(b) amplifying a portion of the human genomic DNA in the sample to produce a calibrant amplicon and an amplicon of the one or more nucleic acid sequences of interest;
(c) melting the amplicons, where in a detector under control of a controller detects fluorescence emissions of each of the amplicons, and the controller uses the fluorescence emissions and corresponding readings of the temperature sensor to determine a melting temperature of each of the amplicons;
(d) causing the controller to compare the determined melting temperature of the calibrant amplicon with an expected melting temperature of the calibrant amplicon;
and
(e) where the determined melting temperature of the calibrant amplicon differs from the expected melting temperature of the calibrant amplicon, causing the controller to determine one or more correction factors and to determine a corrected melting temperature of the amplicon of the one or more nucleic acid sequences of interest.

5 more elements

A method of calibrating a temperature sensor measuring the temperature of a microfluidic channel present in a microfluidic system, the method comprising:

(a) melting one or more calibrant amplicons, wherein the one or more calibrant amplicons are human genomic DNA amplicons amplified by the primers (i) SEQ ID Nos:
7 and 8 or (ii) SEQ ID Nos:
9 and 10 in an amplification reaction comprising a fluorescent dye, wherein a detector under control of a controller detects fluorescence emissions of the one or more calibrant amplicons and the controller uses the fluorescence emissions and corresponding readings of a temperatures sensor to determine a melting temperature of each of the calibrant amplicons;
(b) causing the controller to compare the determined melting temperature of each of the one or more calibrant amplicons with a respective expected melting temperature of each of the one or more calibrant amplicons;
and
(c) causing the controller to determine one or more correction factors and causing the controller to calibrate the temperature of the microfluidic channel detected by the temperature sensor.

3 more elements

Dependencies are inferred from the claim text.

Facts

Application: 13/223,258Filed 31 Aug 2011 · us provisional application
Priority: 31 Aug 2010
Published: US 2012/0178077 A112 Jul 2012
Granted: 17 Mar 2020
Term ends: Not estimatedThe source records don’t support an estimate.

Classification12 codes

G01K 11/06
Measuring temperature based upon physical or chemical changes not covered by groups G01K3/00, G01K5/00, G01K7/00 or G01K9/00: using melting,…
B01L 3/5027
With fluid transport, e.g. in multi-compartment structures: by integrated microfluidic structures, i.e. dimensions of channels and chambers are…
B01L 7/52
Heating or cooling apparatus ; Heat insulating devices: with provision for submitting samples to a predetermined sequence of different…
C12Q 1/6806
Involving nucleic acids: preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay