Multiple displacement amplification on single cell and possible PGD applications

Mol Hum Reprod. 2004 Nov;10(11):847-52. doi: 10.1093/molehr/gah114. Epub 2004 Oct 1.

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.

MeSH terms

  • Alleles
  • DNA / analysis
  • Down Syndrome / diagnosis
  • Female
  • Globins / genetics
  • Heterozygote
  • Humans
  • In Situ Hybridization, Fluorescence
  • Minisatellite Repeats / genetics
  • Mutation / genetics
  • Oligonucleotide Array Sequence Analysis*
  • Polymerase Chain Reaction*
  • Pregnancy
  • Preimplantation Diagnosis
  • Prenatal Diagnosis / methods*
  • Trisomy / diagnosis

Substances

  • Globins
  • DNA