Comprehensive aneuploidy screening in single cells using microarray comparative genomic hybridization methods implications for preimplantation genetic diagnosis2007-09-01 18:13:33
Fertility and Sterility; 2007 Sept; 88(1):S86-87
MR Conces, SL Cook, EB Stelow, RD LeGallo
To develop clinically applicable DNA microarray methods for comprehensive identification of genomic imbalances (ie aneuploidy) in isolated cells.
Array comparative genomic hybridization (array CGH) together with whole genome amplification (WGA) were used to screen for copy number aberrations in single fibroblast cells derived from various aneuploid cell lines.
Materials and Methods:
DNA was extracted from single fibroblasts and amplified using degenerate oligonucleotide primed PCR (DOPPCR). Test and reference DNA were labeled with Cy5 and Cy3 fluorescent dyes, respectively. The labeled DNAs were co-precipitated and hybridized to bacterial artificial chromosome (BAC)-arrays spotted with clones specific for every chromosome at an average resolution of 0.5 megabases. The array was then scanned and Cy5/Cy3 intensity ratios calculated. Ratio values close
to 1 indicate a balanced chromosomal status whereas a ratio >1 or <1 denotes a corresponding gain or a loss of genetic material in the test sample.
DOP-PCR mediated DNA amplification and array CGH permitted reproducible genomic profiling of single cells. Trisomies of chromosomes 13, 15, 16, 18, and 21 along with balanced ploidy levels of the remaining chromosomes were identified in single aneuploid fibroblast cells. Sex mismatched reference DNA was also accurately determined.
Conventional PGD techniques, which rely on fluorescence in situ hybridization (FISH) analysis, usually screen <1/2 of the human chromosomes and consequently many oocytes or embryos carrying lethal abnormalities are not identified. Array CGH together with whole genome amplification allows comprehensive aneuploidy testing and detection of unbalanced translocations in single cells in a manner compatible with transfer during an IVF cycle. Sufficient DNA is also available following amplification to permit testing for single gene defects. This diagnostic may lead to improved IVF outcomes as all aneuploidies can be avoided and could potentially result in a reduction of the multiple birth rate as pregnancy rates could be maintained with fewer embryos transferred.
To Access Article, Click Here