To test the feasibility of our approach, we applied the method to the SNRPN locus (Fig.
We took advantage of this novel assay to simultaneously assess methylation and the copy number status of the SNRPN promoter, thereby allowing the diagnosis and molecular characterization of PWS and AS in <90 min after DNA extraction.
We have developed a methylation-sensitive HRM (MS-HRM) assay that uses the Rotor-Gene 6000 (Corbett Life Sciences) to analyze differential methylation at the SNRPN locus.
The 238-bp SNRPN amplicons containing 21 CpG dinucleotides were melted immediately after PCR.
To investigate whether the effect we observed also depends on the melting temperature of the genomic target, we performed reactions using SNRPN
PCR products as template (1), which resulted in the disappearance of the positional effect.
Multiplex quantitative PCR was used to amplify the FGFR2, KRIT1, and SNRPN genes (FGFR2 forward, CAC AAT CAT TCC TGT GTC GT; FGFR2 reverse, AGC AGT CAA CCA AGA AAA GG; KRIT1 forward, TTC GAA TGG CTA CTT CTA CCT G; KRIT1 reverse, AAA ACG TCT TTT AAA TCA GAG C; SNRPN forward, CTT TGT ACT CCT CCA GCA AC; SNRPN reverse, TAC AGG AAT GAA AGG CAT TA).
Total SNRPN gene copy numbers were calculated by adjusting the relative known doses of the FGFR2 and KRIT1 genes.
After bisulfite treatment, we PCR-amplified a 322-bp fragment of the SNRPN gene promoter region and partial exon 1 corresponding to nucleotides g.
These 5 selected methylation-sensitive probes target the important imprinting region, with 4 probes located on the SNRPN promoter region with PCR fragment sizes of 142, 166, 190, and 247 bp, and 1 probe located on the human necdin-encoding gene (NDN) promoter region (exon 1) with a PCR fragment size of 418 bp.
Here we describe a new diagnostic test for PWS/AS that uses 3 pyrosequencing assays to analyze and quantify 12 CpG sites within the 5' end of the SNRPN
gene dosage was determined by calculating the ratio of net sample signal (NSS; mean sample signal with mean background signal subtracted) from the 3'-SNRPN
assay to that from the 4g25 assay for each sample.
The melt map of a 153-bp genomic DNA region of the SNRPN
CpG island, including 11 CpG dinucleotides, is depicted in Fig.