Determination of copy number differences by amplification
AssigneeFluidigm Corporation
InventorsSimant Dube, Alain Mir, Ramesh Ramakrishnan, Lesley Suzanne Weaver, Bernhard G. Zimmermann
Life of this patent
Estimating the term…
Abstract
The present invention provides for determining relative copy number difference for one or more target nucleic acid sequences between a test sample and a reference sample or reference value derived therefrom. The methods facilitate the detection of copy number differences less than 1.5-fold.
Claims8
An assay method for determining relative copy number difference for one or more target nucleic acid sequences between a test sample and a reference value, the method comprising:
- (a) subjecting at least one test sample, or an aliquot thereof, to preamplification using primers capable of amplifying at least one target nucleic acid sequence to produce a preamplified test sample or aliquot;
- (b) subjecting the preamplified test sample, or an aliquot thereof, to amplification using primers capable of amplifying the target sequence to produce an amplicon, if the target sequence is present in the preamplified test sample or aliquot;
- (c) detecting the amount of amplicon produced by said amplification at one or more selected points during the exponential phase of amplification;
- (d) comparing the amount of amplicon produced at said one or more points with one or more reference values, and determining the relative copy difference for the target sequence, wherein said method can detect a relative copy number difference less than 1.5.
2-8. (canceled)
An assay method for determining relative copy number difference for one or more target nucleic acid sequences between a test sample and a reference sample, the method comprising:
- (a) subjecting at least one test sample, or an aliquot thereof, to preamplification using primers capable of amplifying at least one target nucleic acid sequence and at least one internal control nucleic acid sequence to produce a preamplified test sample or aliquot;
- (b) subjecting a reference sample, or an aliquot thereof, to preamplification using primers capable of amplifying at least one target nucleic acid sequence and at least one internal control nucleic acid sequence to produce a preamplified reference sample or aliquot;
- (c) distributing each of the test and reference samples, or aliquots thereof, into separate compartments of a microfluidic device;
- (d) separately subjecting each of the aliquots to multiplex amplification using primers capable of amplifying the target sequence and primers capable of amplifying the internal control sequence in each aliquot;
- (e) determining threshold cycle (Ct) values for the target sequence and the internal control sequence for each aliquot;
- (f) determining the difference between the Ct value for the target sequence and the Ct value for the internal control sequence (ΔCt value) for each of the test and reference samples;
- (g) determining the difference between the ΔCt value for the test sample and the ΔCt value for the reference sample (ΔΔCt);
- (h) determining the relative copy difference between the test and reference samples for the target sequence;
- wherein said method can detect a relative copy number difference less than 1.5.
10-45. (canceled)
An assay method for determining relative copy number differences for a plurality of target nucleic acid sequences on at least one chromosome in a test sample, as compared to one or more reference values for the target nucleic acid sequences, the method comprising:
- (a) dividing a test sample, or a preamplified test sample, into a plurality of test aliquots or preparing a plurality of test aliquots from aliquots of a preamplified test sample;
- (b) separately subjecting each aliquot to amplification using primers capable of amplifying each target sequence to produce an amplicon, if the target sequence is present in the aliquot;
- (c) detecting the amount of each amplicon produced by said amplification at one or more selected points during the exponential phase of amplification;
- (d) comparing the amount of each amplicon produced at said one or more points with one or more reference values, and determining the relative copy number differences for the target sequences, wherein said method can detect a relative copy number difference less than 1.5.
47-50. (canceled)
An assay method for determining relative copy number differences for a plurality of target nucleic acid sequences on at least one chromosome, between a test sample and a reference sample, the method comprising:
- (a) preparing a plurality of test aliquots and a plurality of reference aliquots from the test and reference samples, or aliquots thereof, or preamplified test and preamplified reference samples, or aliquots thereof, and distributing each aliquot into a separate compartment of a microfluidic device;
- (b) separately subjecting each aliquot to multiplex amplification using primers capable of amplifying at least one of said target sequences and primers capable of amplifying the internal control sequence in each aliquot;
- (c) determining threshold cycle (Ct) values for the target sequence(s) and the internal control sequence for each aliquot;
- (d) determining the difference between the Ct value for each target sequence and the Ct value for the internal control sequence (ΔCt value) for each of the test and reference samples;
- (e) determining the differences between the ΔCt values for the test sample and the ΔCt values for the reference sample (ΔΔCt) for each target sequence;
- (f) determining the relative copy differences between the test and reference samples for each target sequence;
- wherein said method can detect a relative copy number difference less than 1.5.
52-83. (canceled)
Dependencies are inferred from the claim text.