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.
Katz
Mandy G
MG
Centre for Early Human Development, Monash Institute of Reproduction and Development, Monash University, Level 3, 27-31 Wright Street, Clayton 3168, Victoria, Australia. mandy.katz@med.monash.edu.au
Trounson
A O
AO
Cram
D S
DS
D000483:Alleles; D001757:Blastomeres / Q000502:physiology*; D002610:Cheek / Q000196:embryology; D016172:DNA Fingerprinting; D004314:Down Syndrome / Q000196:embryology / Q000235:genetics; D004622:Embryo, Mammalian / Q000166:cytology / Q000502:physiology*; D005260:Female; D005307:Fertilization in Vitro; D005453:Fluorescence; D005784:Gene Amplification; D006801:Humans; D018895:Microsatellite Repeats; D016133:Polymerase Chain Reaction; D016037:Single-Blind Method