Single-cell DNA and FISH analysis for application to preimplantation genetic diagnosis.
The goal of preimplantation genetic diagnosis (PGD) is to avoid transfer of embryos affected with a specific genetic disease or condition. This unit describes the steps involved in amplifying DNA from a single blastomere and specific assays for detecting a variety of DNA mutations. For some assays, whole-genome amplification by primer-extention preamplification (PEP) is performed prior to analysis. Support protocols describe the biopsy of one or two blastomeres from the developing preimplantation embryo, isolation for further investigation of all blastomeres from embryos shown to have the mutant allele, and isolation of single lymphocytes or lymphoblastoid cells as models for single-cell DNA analysis. A procedure for FISH analysis on single interphase blastomeres is provided along with a support protocol for probe validation that is recommended as a preliminary step before performing any PGD FISH analysis.
Chong
Samuel S
SS
Georgetown University Medical Center, Washington, DC, USA.
Gore-Langton
Robert E
RE
Hughes
Mark R
MR
Miron
Patricia Minehart
PM
D001757:Blastomeres / Q000737:chemistry / Q000166:cytology; D004247:DNA / Q000235:genetics* / Q000302:isolation & purification*; D017931:DNA Primers; D005260:Female; D005826:Genetics, Medical; D006801:Humans; D017404:In Situ Hybridization, Fluorescence / Q000379:methods*; D016133:Polymerase Chain Reaction; D011247:Pregnancy; D019836:Preimplantation Diagnosis / Q000379:methods*
D017931:DNA Primers; D004247:DNA