Multiple displacement amplification on single cell and possible PGD applications

Abstract

Multiple displacement amplification (MDA) is a technique used in the amplification of very low amounts of DNA and reported to yield large quantities of high-quality DNA. We used MDA to amplify the whole genome directly from a single cell. The most common techniques used in PGD are PCR and fluorescent in-situ hybridization (FISH). There are many limitations to these techniques including, the number of chromosomes diagnosed for FISH or the quality of DNA issued from a single cell PCR. This report shows, for the first time, use of MDA for single cell whole genome amplification. A total of 16 short tandem repeats (STRs) were amplified successfully with a similar pattern to the genomic DNA. Furthermore, allelic drop out (ADO) derived from MDA was assessed in 40 single cells by analysing (i) heterozygosity for a known beta globin mutation (IVSI-5 C-G) and by studying (ii) the heterozygous loci present in the STRs. ADO turned out to be 10.25% for the beta globin gene sequencing and 5% for the fluorescent PCR analysis of STRs. Moreover, the amplification accuracy of MDA permitted the detection of trisomy 21 on a single cell using comparative genome hybridization-array. Altogether, these data suggest that MDA can be used for single cell molecular karyotyping and the diagnosis of any single gene disorder in PGD.

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

“…4B). The individual marker ADO rates ranged from 0%-33.3%, consistent with previously reported marker ADO rates from similar WGA products (16,17,(27)(28)(29)(30). Thus, the tridecaplex panel demonstrated its versatility for single-cell PGD analysis, either directly or after WGA.…”

“…3 (3, 4 ). HTT alleles are classified based on the number of CAG repeats: normal (Յ26 CAGs), intermediate (27)(28)(29)(30)(31)(32)(33)(34)(35), HD-causing with reduced-penetrance (36 -39 CAGs), and HDcausing with full-penetrance (Ն40 CAGs). Clinical symptoms of HD progress gradually, usually with cognitive and psychiatric manifestations appearing first followed by movement abnormalities and, finally, death (5,6 ).…”

Experience of more than 100 preimplantation genetic diagnosis cycles for monogenetic diseases using whole genome amplification and linkage analysis in a single centre

“…In August 2007, we adopted the platform of WGA for PGD. Such WGA amplifies a major portion of the genome of single cells with good reproducibility, 4 and enables direct mutation detection and linkage analysis simultaneously for accurate determination of the genotype of embryos. We reported our first live birth after PGD for Huntington's disease (HD) using WGA in 2009.…”

Methodology Multiple displacement amplification for preimplantation genetic diagnosis of fragile X syndrome

“…Using the MDA products as a template, we performed only a single round of PCR, which decreases the time required for the PGD program. This strategy is a reliable protocol for obtaining material from a sample with a minuscule amount of DNA, such as a single cell, and several reports have demonstrated high amplification rates (88-100%) with an ADO rate of 7-31% when using MDA-amplified DNA as a template (Handyside et al, 2004;Hellani et al, 2004Hellani et al, , 2005Burlet et al, 2006;Lledo et al, 2007). In particular, Burlet et al (2006) reported successful diagnosis, pregnancy and delivery in the PGD program using MDA for FXS.…”