Patent ID: 8450063

Claim:
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, wherein each test sample is divided into a plurality of replicate aliquots prior to preamplification and/or prior to amplification, and each amplification reaction is carried out in at least two replicate aliquots; (c) detecting the amount of amplicon produced by said amplification at one or more selected points during the exponential phase of amplification in each replicate aliquot; (d) comparing the amount of amplicon produced at said one or more points with one or more reference values, and thereby determining the relative copy difference for the target sequence, wherein said method can detect a relative copy number difference less than 1.5.