Additional considerations in design of singlecell genotyping for PGD using realtime PCR

In clinical PGD, the diagnosis has to be achieved on a single cell, with only one initial PCR reaction possible. In addition, the result has to be available within 24 hours. PCR-based diagnosis of single-cells is susceptible to PCR failure, allele drop-out (when one of the alleles fails to amplify to detectable levels) and contamination. PCR failure means no result, which is undesirable, but allele drop-out and contamination are dangerous as they can lead to an unacceptable misdiagnosis. Thus PCR protocols have to be optimized to minimize PCR failure and allele drop-out, and contamination must be stringently avoided and ideally monitored in each individual sample (by the parallel analysis of hypervariable microsatellite loci) to preclude mis-diagnosis. Finally when confronting a relatively common monogenic disorder such as the P-thalassemias and hemoglobinopathies, ideally the method should be flexible to facilitate detection of a wide spectrum of disease-associated mutation-interactions, precluding the design and standardization of case-dependant protocols each time.

Thus the overall strategy we designed (Vrettou et al., 2004) involved a first-round multiplex PCR (approximate time 1.5 h), containing P-globin gene first-round PCR primers (Setl or Set2) for the amplification of a region of the P-globin gene surrounding the case-specific mutations along with fluorescently-labeled primers for amplification of two polymorphic microsatellite markers GABRB3 (at the GABAA receptor b3 locus in the Angelman/Prader-Willi region on chromosome 15) and D13S314 (Table 17.2). The first-round multiplex PCR was followed by: 1) real-time nested PCR with hybridization probes for analysis of P-globin gene alleles (approximate time 40 min), and in parallel, 2) size analysis of the two microsatellite markers on an automatic sequencer (approximate time 35 min) for monitoring and precluding contamination. Genomic DNA from the parents in each cycle was amplified in parallel along with blank samples from the IVF unit and PCR premix blanks.

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