One-step multiplex polymerase chain reaction for preimplantation genetic diagnosis of Huntington disease

Identification of Novel Microsatellite Markers <1 Mb from the HTT CAG Repeat and Development of a Single-Tube Tridecaplex PCR Panel of Highly Polymorphic Markers for Preimplantation Genetic Diagnosis of Huntington Disease

“…To date, only 10 microsatellite markers have been reported in PGD for HD (11-15, 19, 20 ), with multiplex PCR described for a maximum of 3 markers (14 ). As such, each new PGD case will require testing all available markers and customizing a panel of informative markers specific to each couple.…”

“…Out of the 13 panel markers, 6 have been previously reported and used successfully in PGD (11)(12)(13)(14)(15)19 ). The remaining 7 markers (HD2098407, HD2362117, HD2417179, HD3139793, HD3377975, HD3615631, and HD3829173), each possessing a PIC value Ͼ0.5, are novel.…”

“…The flexibility of amplifying from single cells directly or after WGA demonstrates the versatility of this panel for use in HD PGD, either for linkage-based disease exclusion or as a complement to HTT CAG expansion-mutation detection. Coamplification of the HTT CAG repeat with up to 3 microsatellite markers has been demonstrated (11,14 ). However, coamplification of the CAG repeat with the 13 panel markers appears to be suboptimal probably because of the combined effect of the high GC content of the CAG repeat compared to the other microsatellite markers and the greater number of amplimers in the reaction, resulting in greater amounts of primer dimer and nonspecific PCR products of Յ75 bp that are produced, which overlap with the HTT CAG peaks (data not shown).…”

“…Because different couples may be informative for different markers, the lack of sufficient flanking markers may limit the availability of exclusion PGD to only a subset of at-risk couples. Thus far, only 10 microsatellite markers have been reported in either standard or exclusion PGD (11-15, 19, 20 ), and only a maximum of 3 markers have been coamplified from a single cell (14 ).…”

Experience of Preimplantation Genetic Diagnosis with HLA Matching at the University Hospital Virgen del Rocío in Spain: Technical and Clinical Overview

“…A one-step multiplex single-cell fluorescent PCR is used for the simultaneous amplification of several combinations of markers at the HLA locus alone or in combination with the β -globin locus, using the QIAGEN Multiplex PCR kit (QIAGEN, GmbH; Hilden, Germany) and a protocol previously described [16, 23]. Primer sequences and PCR conditions for HLA-typing in combination with PGD for β -thalassemia have been previously described [16].…”