Patent ID: 6365353
Filing Date: 2002-04-02
Classification: C12Q

Abstract:
A method for determining the relative copy number of nucleic acid sequences of two or more nucleic acid sequences collectives comprising the following steps:a) labeling i) a nucleic acid sequence collective (T) to be investigated; ii) a reference nucleic acid sequence collective (R); and iii) at least one calibration nucleic acid sequence collective (S) having a known deviation in the copy number of its nucleic acid sequences at at least one known position in comparison to the copy number at the corresponding position of the nucleic acid sequences of the reference nucleic acid sequence collective, said calibration nucleic acid sequence collective being substantially identical to the reference nucleic acid sequence collective, wherein each of said nucleic acid sequence collectives i) to iii) is provided with a different label and hybridizing said nucleic acid sequence collectives i) to iii) to a collective of suitable target nucleic acid sequences; b) detecting at each position of said nucleic acid sequences the intensity of the labeling of each of said differently labeled nucleic acid sequence collectives i) to iii) being hybridized with said target nucleic acid sequences; c) determining for said each position a quotient QR by dividing the intensity of the labeling of the nucleic acid sequences of said nucleic acid sequence collective to be investigated and the corresponding intensity of the labeling of the nucleic acid sequences of said reference nucleic acid sequence collective: d) determining for said each position a quotient QS by dividing the intensity of the labeling of the nucleic acid sequences of said nucleic acid sequence collective to be investigated and the corresponding intensity of the labeling of the nucleic acid sequences of said calibration nucleic acid sequence collective; e) determining uncorrected relative copy numbers of the nucleic acid sequences of said nucleic acid sequence collective to be investigated in comparison to the nucleic acid sequences of said reference nucleic acid sequence collective by means of standardizing the quotients QR obtained in step c) by dividing the value of QR at each position and the value of QR which occurs most frequently over all positions; f) determining uncorrected relative copy numbers of the nucleic acid sequences of said nucleic acid sequence collective to be investigated in comparison to the nucleic acid sequences of said calibration nucleic acid sequence collective by means of standardizing the quotients QS obtained in step d) by dividing the value of QS at each position through the value of QS which occurs most frequently over all positions; g) determining at least two anchor positions in the nucleic acid sequences of said calibration nucleic acid sequence collective and said reference nucleic acid sequence collective showing a deviation in the amount of the uncorrected relative copy number being equal to said known deviation in the copy number of the nucleic acid sequences of the calibration nucleic acid sequence collective at at least said known position in comparison to the copy number at the corresponding position of the nucleic acid sequences of the reference nucleic acid sequence collective; h) calculating a correction curve using the deviations between the uncorrected relative copy number at said anchor positions obtained in step g) and corresponding uncorrected relative copy number which are expected due to said known deviation in the copy number of the nucleic acid sequences of the calibration nucleic acid sequence collective in comparison to the copy number of the nucleic acid sequences of the reference nucleic acid sequence collective; and, i) determining final relative copy numbers of the nucleic acid sequences of said nucleic acid sequence collective to be investigated by correcting said uncorrected relative copy numbers obtained in step e) with the aid of said correction curve obtained in step h); wherein said nucleic acid sequence collectives are selected from the group consisting of mRNA, cDNA, genomic DNA, and-chromosomal DNA.