Chapter 15 - Preimplantation genetic diagnosis for monogenic disorders: multiplex PCR and whole-genome amplification for gene analysis at the single cell level

Summary

The first preimplantation genetic diagnosis (PGD) for monogenic disease was polymerase chain reaction- (PCR-) based. Contamination with extraneous DNA was recognized very early on after the development of PCR as a cause of erroneous diagnosis. Minisequencing is a method in which one fluorescent dideoxynucleotide is inserted at the site of the mutation. The European Society for Human Reproduction and Embryology (ESHRE) PGD Consortium guidelines recommend that at least 50 single cells and 50 blanks are tested to have an accurate idea of the allele drop-out (ADO) and contamination levels. Methods of whole-genome amplification (WGA) could overcome the difficulty of insufficient DNA, providing sufficient template for numerous genetic analyses, such as array comparative genomic hybridization (array- CGH), microsatellite analysis, or single nucleotide polymorphism (SNP) analysis. A non-PCR-based isothermal method, termed multiple displacement amplification (MDA), has been applied to DNA samples, leading to the synthesis of DNA with limited sequence representation bias.