Strategies for preimplantation genetic diagnosis of single gene disorders by DNA amplification

Abstract

Preimplantation genetic diagnosis (PGD) is an alternative to prenatal diagnosis for ensuring the genetic health of offspring born to families affected by inherited disease. This paper sets out to review current protocols for the diagnosis of single gene defects in human preimplantation embryos. These methods, which depend on DNA amplification using PCR, are subject to a vareity of pitfalls, such as allele dropout (ADO), contamination and reduced amplification efficiency. Advances in single cell DNA amplification, such as improved multiplex PCR protocols, fluorescent‐PCR and whole genome amplification (WGA), can be applied to address some of these problems. Different PGD strategies are discussed in the context of their clinical application. Copyright © 1998 John Wiley & Sons, Ltd.

Molecular Diagnostics in Preimplantation Genetic Diagnosis

“…As with reduced amplification efficiencies, increased ADO is noted in degenerating cells presumably the result of strand-specific DNA degradation. 52 Additionally, access to the target genomic sequence by the primers and Taq polymerase may be restricted by, for example, adjacent G/C rich regions reducing denaturation efficiency or differing degrees of folding perhaps related to the stage of the cell cycle. 27 Whatever the exact cause, ADO likely arises in the initial cycles of the primary PCR before the number of target molecules is increased by the process of amplification.…”

Long-Chain L-3-Hydroxyacyl-Coenzyme A Dehydrogenase Deficiency: A Molecular and Biochemical Review

“…An emerging alternative strategy that avoids termination of pregnancy is preimplantation diagnosis (Wells and Sherlock, 1998) using cells from in vitro fertilization-generated embryos. This technique has recently been applied for diagnosis of MCAD deficiency (Ioulianos et al, 2000;Sermon et al, 2000).…”

Application of Hot Start PCR Method in PCR-based Preimplantation Genetic Diagnosis

“…PGD for monogenic diseases is susceptible to problems of contamination, PCR failure, and most significantly, ADO, which may lead to misdiagnosis. 14,15) We take about three to six months to set up a PCR condition, and it was tested for DNA dilution, single lymphocyte and some blasto meres. It was mostly difficult in that time of diverting from a single level of DNA to a single cell.…”