Developing and Optimizing Conditions for Single Cell Genetic Analysis
Preimplantation Genetic Diagnosis (PGD) allows for couples to obtain genetic information at the embryo stage, therefore preventing lethal genetic diseases in their offspring. A recent multi-center study determined a 17% amplification failure rate overall and a 3.4% misdiagnosis rate (ESHRE PGD consortium steering committee, 2002). Experiments presented here were conducted to decrease those rates by optimizing single cell sensitivity and specificity of PGD techniques. Primer extension preamplification (PEP) was used after initial amplification failure, followed by gene specific PCR. Reamplification experiments yielded 54% amplification rates using blastomeres that previously failed amplification. In the clinical trial, four of six blastomeres were diagnosed that had previous amplification failure. PEP and subsequent PCR testing is a useful means to diagnose samples that previously failed amplification. Different lysis buffers/techniques were used to determine the amplification efficiencies of heterozygous single cells (n = 100/group). Amplification rates were 38% using Liquid Nitrogen, 97% using Potassium hydroxide/dithiothreitol (KOH), 41% by boiling, and 85% for water. Allele drop-out was minimal in the KOH group, but was high in the water group. KOH lysis is superior in both sensitivity and specificity to other lysis methods tested. Nuclear and cytoplasmic morphology of biopsied blastomeres was evaluated and correlated with subsequent PCR amplification efficiencies. Cells with a visible nucleus had 100% amplification versus 36% in cells that did not, regardless of the cytoplasmic integrity. Nuclear evaluation studies demonstrate the importance of determining nuclear integrity of biopsied cells. Multiple displacement amplification (MDA) was used to generate sufficient DNA for eventual use in array comparative genomic hybridization (CGH) to determine chromosome complement. Sporadic MDA results occurred at the single cell level; larger cell quantities consistently amplified. MDA can generate sufficient quantities of DNA for array CGH. Preliminary results show promise; further optimization will be required to ascertain the use of array CGH. These studies provide optimization of single cell sensitivity/specificity and lay the foundation for further studies in single cell diagnosis. It is anticipated that an entire array of gene defects and chromosomal conditions can be diagnosed by PGD in the future prevention of severe human disease.
Genetic analysis; Preimplantation genetic diagnosis; Single-cell, Cell Biology, Genetics
Networked Digital Library of Theses and Dissertations