Simultaneous Application of Chromosomal Microarray Analysis and Polymerase Chain Reaction Genetic Disease Detection
Date
2014-10-07
Authors
Tran, Khoa D
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Abstract
This thesis demonstrates the successful integration and application of chromosomal microarray analysis (CMA) and mutation-specific polymerase chain reaction (PCR) within 24 hours for the detection of unaffected euploid embryos from couples at risk for genetic disease undergoing in vitro fertilization treatment. Currently, array comparative genomic hybridization (aCGH) for detecting aneuploidy and PCR, and/or sequencing, for mutation status detection are performed at two different time points, thus, not appropriate for delivering results within a time frame suitable for a fresh embryo transfer. The techniques presented here have been optimized for analysis and reporting within a 24-hr time frame, allowing clinicians to offer their patients the option of fresh embryo transfer. Whole genome amplification (WGA) of trophectoderm embryo biopsy was performed and aliquots of WGA product were divided for CMA and for laboratory-developed fluorescent PCR coupled with capillary electrophoresis for genetic disease diagnosis. The combined protocol requires no modification of the CMA protocol and no pre-amplification steps were required prior to WGA. The WGA products were subjected to single round PCR testing for mutational and linked marker detection, except for mutations involving triplet repeats where nested PCR was necessary. Although current techniques are available for detecting aneuploidy and genetic mutation status, such as aCGH or SNP arrays for aneuploidy and PCR or sequencing for gene mutation, these methods cannot be combined for analysis within a 24-hour time frame. The successful integration and application of these important techniques provides a comprehensive diagnostic approach that can be offered to couples at risk for single-gene disorders wishing to receive a fresh embryo transfer. This new approach allows laboratories currently equipped to perform aCGH and PCR to utilize their existing setup for a novel comprehensive diagnostic protocol without additional equipment acquisition.
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Keywords
Chromosomal Microarray Analysis, Genetic disease mutation, Fertility, PGD