Multiple displacement amplification (MDA) a new approach for preimplantation genetic diagnosis (PGD): our experience

Abstract

For single-gene disorders, the development and validation of highly sensitive amplification strategies are required, often using nested-PCR, whole-genome amplification (WGA), or fluorescent PCR methods. There is a need for a technique that would be able to amplify the single-cell DNA with a high fidelity that suits the diagnosis of any known single-gene disorder by standard PCR. MDA is a technique used in the amplification of very low DNA quantities in clinical samples. We investigate the use of MDA from single cells as a universal first step for PGD of single defects. Whole genome from single cells was succesfully amplified using MDA. MDA products were used as template for PCR reactions of different locus involved in monogenic diseases: CAG expansion in IT15 gene (Huntington Disease), 5'-CGG expansion in FMR1 gene (X-fragile syndrome), CTG expansion in DMPK gene (Myotonic Dystrophy) and CAG expansion in MJD1 gene (Machado-Joseph disease). In order to detect the homogeneity of the amplification across the genomic target and the value in PGD, MDA products were used for the amplification of 16 STR used in the diagnosis of 8 monogenic diseases by segregation studies: Duchenne dystrophy, Marfan syndrome, X-linked Retinoschisis, Acute intermittent porphyria, Myotonic Dystrophy, Incontinentia pigmenti, Adrenoleukodistrophy, Emerrin-Dreiffus. MDA was successful in 4/4 single lymphocytes. The determination of the quality of the amplified DNA showed similar to genomic DNA. PCR amplification from the single lymphocyte DNA was detected in 252/252 reactions. Allele sizes matched those amplified from genomic DNA. At heterozygous loci, preferential amplification of alleles was reported and allele drop-out (ADO) was detected in 15/252 (6%). The MDA product analysis showed significant consistency in the quality and efficiency of MDA amplification. The major disadvantage of PCR–WGA methods is the slippage of microsatellites. We have illustrated an efficient and reliable method for diagnosis at the single-cell level, using standard PCR procedures. The MDA test would become a universal first step in PGD, since enough DNA from a single cell to allow for multiple PCR analyses. At the same time, high amplification efficiency and low ADO rates are seen compared with nested multiplex PCR. According to our results, MDA is the method of choice for PGD cycles.