DNA fingerprinting of sister blastomeres from human IVF embryos.

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.