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Single-cell Comparative Genomic Hybridization and Its Application in Preimplantation Genetic Screening

Author: TanZuo
Tutor: TanYueQiu
School: Central South University
Course: Genetics
Keywords: whole genome amplification comparative genomic hybridization embryo aneuploidy preimplantation genetic screening
CLC: Q78
Type: Master's thesis
Year: 2010
Downloads: 69
Quote: 0
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Abstract


Objective:Chromosomal aneuploidy is the major reason of embryonic development abnormalities, spontaneous abortion and congenital birth defect. Preimplantation genetic screening (PGS) refers to procedures that are performed on embryos prior to implantation, PGS is used to identify the embryo with chromosomal aneuploidy which lead to birth defect, and also provided as a laboratory technology to locate the mutant region and understand the mechanism of specific chromosome disease. Traditional PGS which is based on fluorescence in situ hybridization (FISH) only detect limited chromosomes and the procedure is time-consuming. Establish a high-flux single-cell comparative genomic hybridization (CGH) for PGS will benefit clinical diagnosis and basic research.Methods:Clinical single cell samples were collected for whole genomic amplification(WGA) by combining primer extension preamplification(PEP) with degenerate oligonucleotide primed-PCR (DOP-PCR) technology after karyotype analysis, Red fluorescence tag were labeled during amplification, After mixed with the standard normal male DNA which marked green fluorescence, CGH analyze was performed follow standard protocol.Single-cell CGH technology has established by comparative karyotype analysis. Different cell types including dysembryoplasia embryos and the trophectoderm of preimplantation blastocyst were collected and tested in this system, statistic analysis were employed to discuss diagnose reliability and practicality of this new method.Results:16 single-cell whole genomes DNA were effectively amplified and reflected correct chromosome copy number when applied in CGH assay. Compared with traditional DOP-PCR, PEP-DOP-PCR method, PEP-DOP-PCR combined CGH technology is more stable and suitable for single-cell CGH.Chromosome copy number abnormality has been successful detected in 23 embryos which have development abnormalities. Phenomenon of cleavage embryos exist majority chromosome aneuploidy and mosaic were confirmed in followed FISH analysis.17 preimplantation blastocyst were analyzed by single-cell CGH, blastula stage has lower rate of chromosome abnormality than cleavage embryos;Trophectoderm analysis found one embryo with trisome 21 in accord with the G banding karyotype result of this stem cell line.Conclusions:Reliable single-cell CGH technology has been established by PEP-DOP-PCR combined CGH. The application of this technology has confirmed that aneuploidy is one of the most important facts of embryo development abnormalities.The existence of embryo mosaic is confirmed in our new technology system. The successful application in blastula biopsy of our single-cell CGH demonstrate application prospects of this new technology in clinical PGS.The confirmation with karyotype of hESCs indicates a new strategy to screen specific karyotype hESCs.

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CLC: > Biological Sciences > Molecular Biology > Genetic engineering (genetic engineering)
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