Photocrosslinking oligonucleotide hybridization assay for concurrent gene dosage and CpG methylation analysis.

The phenotypic effects of aberrant gene expression are indistinguishable, regardless of whether the underlying mutation is one of gene copy number (deletion or duplication) or modification of differentially methylated CpG sites occurring in critical regulatory sequences in gene promoters. The XLnt photocrosslinking oligonucleotide technology provides for the hybridization-dependent covalent attachment of probe to target, allowing survival of the probe/target complex under otherwise denaturing conditions. Posthybridization wash stringency can be substantially higher than under standard techniques, leading to a marked increase in signal-to-noise ratios. In addition, the reduction in nonspecific background provides linearity to XLnt-based oligonucleotide assays comparable to that otherwise achieved only with very long probes. The technology is thus ideally suited for combining the high-throughput capacity of oligonucleotide hybridization platforms with accurate measurement of relative gene dosage. By integrating the XLnt system with an assay design separating probe/target immobilization and signal elaboration functions, relative gene dosage assessment can be applied to the quantitation of fractional resistance to methylation-sensitive restriction enzyme (MSRE) digestion. The method described below provides for the development of photocrosslinking oligonucleotide assays for relative gene dosage and fractional locus CpG methylation in a microtiter plate-based format.

High-throughput detection of submicroscopic deletions and methylation status at 15q11-q13 by a photo-cross-linking oligonucleotide hybridization assay.

BACKGROUND: Current technologies for assessing genetic deletions and duplications of greater than one kilobase are labor-intensive or rely on PCR-based methods, and none offers the ability to simultaneously detect dosage abnormalities, assess 5'-to-3' cytosine-guanosine (CpG) methylation, and interrogate single-nucleotide polymorphisms (SNPs). We describe a high-throughput platform for direct gene-dosage determination capable of concurrent assessment of other forms of gene modification. METHODS: We used a light-activated interstrand nucleic acid cross-linking system (XLnt technology) to determine gene dosage at the 15q11-q13 deletion/duplication locus. We incorporated restriction enzyme digestion of genomic DNA into the method to assess CpG methylation in parallel with gene dosage. For method validation we used DNA from 31 cell lines with previously characterized 15q11-q13 gene dosage and parental origin status. Diagnostic cutoffs were set at 0.5 +/- 0.15, 1 +/- 0.15-0.25, and 2 +/- 0.3. RESULTS: Dosage-only experiments showed discrimination of deletions (n = 21) from healthy controls (NCs; n = 27) in all samples. Five of 49 samples gave results outside of specification. Concurrent evaluation of dosage and CpG methylation yielded dosage results within specification for 18 of 19 deletion and 8 of 12 NC samples. Paternal deletion and NC methylation pattern results were within specification in 17 of 19 and 9 of 12 runs, respectively. No overlap was demonstrated between value sets for the two groups. CONCLUSIONS: The XLnt technology provides a rapid, high-throughput platform for the accurate determination of gene dosage. The flexibility of this technology allows parallel interrogation of gene dosage, CpG methylation, and SNPs.