Archival fixed and analysed preimplantation human embryonic cells: a DNA resource for retrospective PCR analysis at the cellular level |
| |
Authors: | Smith TA; Wall MB; Muggleton-Harris AL |
| |
Institution: | Division of Obstetrics and Gynecology, UMDS, St Thomas's Hospital, London, UK. |
| |
Abstract: | Biopsies of human embryonic cell preparations previously analysed by
cytogenetic and/or fluorescent in-situ hybridization (FISH) chromosome
probes provide a unique reference DNA resource for the archival
preimplantation genetic diagnosis (PGD) of the transferred embryo. DNA
polymerase chain reaction (PCR) may be utilized on these fixed cell
preparations to verify equivocal FISH/PGD results. Retrospective PCR
screens of the genotype of biopsied embryonic cell(s) may be of benefit in
the case of a suspected genetic mutation. Currently, carrier detection or
linkage analysis is often not possible because of early death of the fetus,
or of patients with a lethal disease. Alternatively, fixed/stained 'failed
fertilized' oocytes provide a resource to extend genetic analysis to
infertile patients. A successful research is described which minimizes loss
of individual analysed fixed/stained oocytes, metaphase chromosomes, and
embryonic cell samples. Initial DNA amplification takes place in situ using
a modified PCR protocol. Comparative cellular studies using primer sets
previously used for PGD analyses show that 65% of the preparations
amplified unequivocally using the modified protocol and primers for a CA
repeat motif gene sequence, in comparison with 81% using the original PCR
protocol. With further refinement and optimization, the methods outlined
have the potential to retrospectively screen archival fixed chromosomes,
gametes, and embryonic cells for clinical application, and enable the
further study of the fixed human preimplantation embryo at the
morphological, cell and molecular level.
|
| |
Keywords: | |
本文献已被 Oxford 等数据库收录! |
|