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Single-cell analysis of unstable genes

  • Papers From The International Working Group On Preimplantation Genetics
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Abstract

Purpose: We have developed sensitive diagnostic procedures for studies on the normal and mutant alleles of the triplet repeat genes associated with myotonic dystrophy and fragile X in single human somatic cells, gametes and embryos.

Methods: Polymerase chain reaction (PCR) assays for the normal alleles of the myotonic dystrophy and fragile X loci have been refined to the sensitivity of the single cell. In addition, we have developed a simple PCR-based technique, termed ‘Repeat Primer PCR’, which can detect the full fragile X expansion in small samples of buccal cells.

Conclusions: The assay for the triplet repeat sequence in the myotonic dystrophy locus could not be used to study stability since we observed additional PCR products derived fromin vitro expansion of the triplet repeat sequence during the PCR reaction itself. The implications ofin vitro expansion and allele drop-out for studies on the timing of the expansion in development and preimplantation diagnosis of triplet repeat diseases are discussed. The development of a new PCR procedure to identify the expanded alleles of the fragile X locus could prove invaluable for monitoring the timing of repeat expansion in early embryonic development. Triplet repeat polymorphisms provide a means of identifying the maternally and paternally-derived alleles of the myotonic dystrophy gene. Using single cell reverse transcriptase PCR analysis, we have monitored the onset of the myotonic dystrophy gene transcription in early preimplantation embryos. Transcripts from the paternally- inherited allele of the myotonic dystrophy gene are already detectable in the 1-cell stage human embryo.

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Author notes

  1. C. Holding

    Present address: Laboratory of Human Molecular Genetics, Downing Street, CB2 3EH, Cambridge, UK

  2. E. Kontogianni

    Present address: Centre for Reproductive Fertility and Genetics, Leof. Kifisias 282, 15232, Halandri, Athens, Greece

Authors and Affiliations

  1. Molecular Embryology Unit, Institute of Child Health, 30 Guilford Street, WC1N 1EH, London, UK

    R. Daniels, C. Holding, E. Kontogianni & M. Monk

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  1. R. Daniels
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  2. C. Holding
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  3. E. Kontogianni
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  4. M. Monk
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Daniels, R., Holding, C., Kontogianni, E. et al. Single-cell analysis of unstable genes. J Assist Reprod Genet 13, 163–169 (1996). https://doi.org/10.1007/BF02072539

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  • DOI: https://doi.org/10.1007/BF02072539

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