Probes and methods for determining the presence or absence of genetic segments

摘要

A method for determining the presence or absence of a genetic segment of interest, such as an exon, an intron or a promoter, in a DNA-containing sample, and probe sets for use in such methods, including probe sets comprising oligonucleotide probes having nucleotides sequences selected from those of SEQ ID NOS: 1-101.

主权项

1. A method for determining the presence or absence of a genetic segment of interest in a DNA-containing sample, the method comprising:
(i) bringing at least a first probe set comprising a plurality of replicates of at least one oligonucleotide probe that interrogates a first cognate sequence within said segment of interest into contact with (a) a plurality of reference samples in which the genetic segment of interest is absent, and (b) a plurality of reference samples in which the genetic segment of interest is present, under conditions that allow probe-cognate sequence hybridisation to occur; (ii) measuring the intensity of probe-sample hybridisation of each of the reference samples, thereby obtaining a first cluster of hybridisation intensity values for the reference samples in which the genetic segment of interest is absent and a second cluster of hybridisation intensity values for the reference samples in which the genetic segment of interest is present; and (iii) establishing a “no call” region of hybridisation intensity values that lies in the region between said first and second clusters and which is bounded by a lower no call boundary (“LNC”) and an upper no call boundary (“UNC”), wherein the LNC and UNC represent statistical confidence limits for assigning a hybridisation intensity value to said first and said second clusters, respectively; iv) contacting the first probe set with at least one DNA-containing test sample under conditions that allow probe-cognate sequence hybridisation to occur; v) measuring the intensity of probe-sample hybridisation of the at least one test sample; and vi) comparing the measured hybridisation intensity with the LNC and the UNC, wherein a measured hybridisation intensity below the LNC indicates that said genetic segment of interest is absent in the test sample and a measured hybridisation intensity above UNC indicates that said genetic segment of interest is present in the test sample; wherein: the DNA of the reference samples and/or the test sample has been labelled with a fluorescent label and wherein measuring hybridisation intensity comprises measuring the fluorescence signal of the fluorescent label at each oligonucleotide probe location; the hybridisation intensity for each probe set is measured as I/B, where:
I is determined as a measure of central tendency of the measured fluorescence signal of the replicates of each oligonucleotide probe; andB is determined as a measure of central tendency of the background fluorescence signal of the replicates of each oligonucleotide probe; and the LNC and UNC values are calculated according to the following formulae:LNC=TI/B·[2·a⁡(I/B)n-aMedian⁢⁢I/B]-a⁡(I/B)n2TI/B-aMedian⁢⁢I/BUNC=TI/B·[pMedian⁢⁢I/B-2·p⁡(I/B)1]+p⁡(I/B)12pMedian⁢⁢I/B-TI/Bwhere⁢:TI/B=pMedian⁢⁢I/B·a⁡(I/B)n-aMedian⁢⁢I/B·p⁡(I/B)1pMedian⁢⁢I/B+a⁡(I/B)n-aMedian⁢⁢I/B-p⁡(I/B)1 aMedianI/B is a measure of central tendency of the measured I/B values of the reference samples in which the genetic segment of interest is absent; pMedianI/B is a measure of central tendency of the measured I/B values of the reference samples in which the genetic segment of interest is present; a(I/B)n is the greatest measured I/B value of the reference samples in which the genetic segment of interest is absent; and p(I/B)1 is the lowest measured I/B value of the reference samples in which the genetic segment of interest is present.