Measurement of locus copy number by hybridisation with amplifiable probes - PubMed (original) (raw)

Measurement of locus copy number by hybridisation with amplifiable probes

J A Armour et al. Nucleic Acids Res. 2000.

Abstract

Despite its fundamental importance in genome analysis, it is only recently that systematic approaches have been developed to assess copy number at specific genetic loci, or to examine genomic DNA for submicro-scopic deletions of unknown location. In this report we show that short probes can be recovered and amplified quantitatively following hybridisation to genomic DNA. This simple observation forms the basis of a new approach to determining locus copy number in complex genomes. The power and specificity of multiplex amplifiable probe hybridisation is demonstrated by the simultaneous assessment of copy number at a set of 40 human loci, including detection of deletions causing Duchenne muscular dystrophy and Prader-Willi/Angelman syndromes. Assembly of other probe sets will allow novel, technically simple approaches to a wide variety of genetic analyses, including the potential for extension to high resolution genome-wide screens for deletions and amplifications.

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Figures

Figure 1

Figure 1

General principle of MAPH. Test DNA is denatured and hybridised with a set of amplifiable probes, each recognising a unique region in the genome. After stringent washing to allow retention only of specifically bound probes, the bound probe fragments are amplified using a common primer pair and quantified, for example after polyacrylamide gel electrophoresis. Since an excess of probe is used, the amount of each probe recovered will be proportional to the copy number of the corresponding sequence in the test DNA.

Figure 2

Figure 2

Gel analysis of recovered probes from analysis of DNA from 10 individuals examined using a mix of 40 probes from human loci, of which 13 are shown in the enlarged region. The chromosome or locus of origin for each probe is shown on the right. Arrows mark deleted bands (see also Fig. 3): individual 9 is a male with a complete deletion of exon 53 of DMD, and heterozygous deletions of this region of DMD can be seen in females 2 and 7 as reductions in the intensity relative to the adjacent Xp22 probe. Individuals 1 and 6 have heterozygous deletions of SNRPN.

Figure 3

Figure 3

Quantitative analysis of signal intensity from two separate experiments each examining 12 DNA samples (the 10 individuals shown in Fig. 2 plus two further control samples) with a total of 40 probes. Phosphorimager data were used to produce estimates of relative intensity for each band, with the mean for each probe standardised to 1.0 (see Materials and Methods). Among control samples, ratios centre on 1.0 for autosomal probes and X-linked probes in females, and are clustered around 0.5 for X-linked probes in males.

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