Embryology. DNA fingerprinting of sister blastomeres from human IVF embryos
Authors: Katz, M.G.; Trounson, A.O.; Cram, D.S.
Source: Human Reproduction, Volume 17, Number 3, March 2002 , pp. 752-759(8)
Publisher: Oxford University Press
Abstract: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.
Document Type: Original Article
Affiliations: Centre for Early Human Development, Monash Institute of Reproduction and Development, Monash University, Clayton, Victoria and
Publication date: 2002-03-01
- Human Reproduction features full-length, peer-reviewed papers reporting original research, clinical case histories, as well as opinions and debates on topical issues. Papers published cover the scientific and medical aspects of reproductive physiology and pathology, endocrinology, andrology, gonad function, gametogenesis, fertilization, embryo development, implantation, pregnancy, genetics, genetic diagnosis, oncology, infectious disease, surgery, contraception, infertility treatment, psychology, ethics and social issues. The highest scientific and editorial standard is maintained throughout the journal along with a rapid rate of publication.