Molecular Human Reproduction Vol. 1, NUMBER 2 pp. 101-109, 1995
© European Society of Human Reproduction and Embryology 1995
research-article |
Reliability and accuracy of polymerase chain reaction amplification of two unique target sequences from biopsies of cleavage-stage and blastocyst-stage human embryos
1Department of Anatomy, University of Cambridge Downing Street, Cambridge CB3 8TE 2Department of Obstetrics and Gynaecology Sixth floor, North Wing, St Thomas's Hospital, Lambeth Palace Road, London SE2 7EH, UK
To whom correspondence should be addressed at: 3To whom correspondence should be addressed at: Zeneca Pharmaceuticals, Infection Department, 14.30, Alderley Park, Macclesfield, Cheshire SK10 4TG, UK
Human embryos have been biopsied at either the cleavage or the blastocyst stage of development. One to two blastomeres were removed from cleavage-stage embryos and 2–6 cells from blastocysts. The biopsy specimens were subjected to gene amplification by the polymerase chain reaction (PCR) and a comparison made of amplification efficiencies of two unique target sequences, one located within the ß-globin gene and containing the sickle-cell locus and the other a polymorphic dinucleotide repeat. When the cleavage-stage biopsy sample consisted of an intact blastomere with a clearly discernible nucleus, an amplification efficiency of 89% was achieved for each target locus. This was similar to that achieved with cleavage-stage biopsy samples consisting of two blastomeres or with blastocyst biopsy samples consisting of 2–3 trophectoderm cells. When biopsy samples consisted of four or more trophectoderm cells, both target loci were amplified in all samples tested. When the biopsy sample was heterozygous at the dinucleotide repeat locus and the biopsy consisted of one or more intact cells with a clearly discernible nucleus, both alleles were amplified in >80% of biopsy samples. When four or more trophectoderm cells were used for the PCR, both alleles were amplified in all heterozygous samples. Target sequences were never amplified from biopsy samples which lysed prior to transfer into the reaction tube. Analysis of DNA fragments amplified from the dinucleotide repeat locus indicated that in most cases faithful amplification of biopsy DNA template had taken place. However, in one case, fragments were identified which could not have resulted from the amplification of embryonic sequences alone, indicating that contamination with extraneous DNA may have taken place. The significance of this finding for therapeutic preimplantation genetic diagnosis is discussed.
ß-globin/CA repeat/embryo biopsy/polymerase chain reaction