Genotyping Efficiency of 2 Primer Sets and an Unlabeled Oligonucleotide Probe for the p.Phe508del in Exon 10 of the CFTR Gene as Determined with High-Resolution Melting Analysis

Single-cell high resolution melting analysis: A novel, generic, pre-implantation genetic diagnosis (PGD) method applied to cystic fibrosis (HRMA CF-PGD)

“…This could be required either in cases where one of the partners is affectedand the other is a carrier of a different CF-causing mutationor to allow simultaneous amplification of a known mutation-linked polymorphism, in order to provide additional evidence for the presence or absence of the mutation to enhance diagnostic accuracy (it is worth noting that during workup of the proposed methodology, up to 8 different primer combinations, aside from the STRs, could be simultaneously amplified in the same first PCR conditions. iii) Our laboratory has accumulated significant experience in assigning melting profiles to genotype status following HRM analysis of these amplicons in routine carrier screening and CF prenatal diagnosis [14,17]; and iv) the primers have been extensively tested and can be co-amplified without giving by-products that could interfere with genotyping. Only the forward primer for exon 17b amplicon 1 was redesigned in order to include in the PCR product the mutation c.3140-26ANG.…”

Prenatal diagnosis for CF using High Resolution Melting Analysis and simultaneous haplotype analysis through QF-PCR

“…Exon 11 also includes a lot of non-disease causing polymorphisms which in combination with p.Phe508del can result in misinterpretation. For this reason, in order to detect it specifically, in the second PCR reaction we also used an unlabelled probe genotyping assay as previously described [12]. The conditions and cycling for the asymmetric 2nd round PCR for exon 11 are shown in Supplementary data Tables 6 & 7.…”