Human Reproduction, Vol. 17, No. 3, 752-759,
March 2002
© 2002 European Society of Human Reproduction and Embryology
DNA fingerprinting of sister blastomeres from human IVF embryos
1 Centre for Early Human Development, Monash Institute of Reproduction and Development, Monash University, Clayton, Victoria and 2 Monash IVF, Melbourne, Australia
BACKGROUND: Previously published single cell DNA fingerprinting systems have been plagued by high rates of allele drop-out (ADO) and preferential amplification (PA) preventing clinical application in preimplantation genetic diagnosis. METHODS: Tetranucleotide microsatellite markers with high heterozygosity, known allelic size ranges and minimal PCR stutter artefacts were selected for chromosomes X, 13, 18 and 21 and optimized in a multiplex fluorescent (FL)-PCR format. FL-PCR products were analysed using the ABI Prism 377 DNA sequenator and Genescan software. Validation of the DNA fingerprinting system was performed on single diploid (n = 50) and aneuploid (n = 25) buccal cells and embryonic blastomeres (n = 21). RESULTS: The optimized pentaplex PCR DNA fingerprinting system displayed a high proportion of successful amplifications (>91%) and low ADO and PA (<6%) when assessed on 50 human buccal cells. DNA fingerprints of single cells from a subject with Down's syndrome detected the expected tri-allelic pattern for the chromosome 21 marker, confirming trisomy 21. In a blind study on 21 single blastomeres, all embryos were identifiable by their unique DNA fingerprints and shared parental alleles. CONCLUSIONS: A highly specific multiplex FL-PCR based on the amplification of five highly polymorphic microsatellite markers was developed for single cells. This finding paves the way for the development of a more complex PCR DNA fingerprinting system to assess aneuploidy and single gene mutations in IVF embryos from couples at genetic risk.
Key words: aneuploidy/DNA fingerprinting/embryonic blastomeres/single cell PCR
3 To whom correspondence should be addressed at: Centre for Early Human Development, Monash Institute of Reproduction and Development, Level 3, 27–31 Wright St, Clayton 3168, Victoria, Australia. E-mail: mandy.katz{at}med.monash.edu.au
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