Amplifying Complex Sequences
Asuragen scientists overcame a 20-year challenge by developing a novel technology for reliably amplifying hard-to-sequence, GC-rich regions and other highly repetitive DNA sequences.
HIGH-RESOLUTION AMPLIFICATION AND GENOTYPING TECHNOLOGIES FOR PREIMPLANTATION GENETIC DIAGNOSIS OF FRAGILE X SYNDROME FROM SINGLE CELLS
Stela Filipovic-Sadic1, Karen Handschuh2, Kangpu Xu2, and Gary J Latham1
1Asuragen, Inc., Austin, TX; 2Center for Reproductive Medicine, Weill Cornell Medical College, New York, NY
Introduction: Preimplantation genetic diagnosis (PGD) methods are increasingly used to detect chromosomal abnormalities and genetic disorders relevant to in vitro fertilization (IVF). One such disorder is fragile X syndrome (FXS), the most common form of inherited intellectual disability. An estimated 1.5 million women in the US are fragile X carriers yet most are unaware of their carrier status. Currently, the identification of FXS by PGD is limited to low-resolution linkage analyses. The goal of this study was to assess the performance and reproducibility of a new high-resolution method for fragile X PGD. The study utilizes a set of well-characterized lymphoblastoid fragile X cell lines as a model for the low cell count biopsies used in PGD.
Methods: Lymphoblastoid cells from 5 fragile X cell lines (one normal, two premutations (PM), and two full mutations (FM)), along with matching genomic DNA samples, were genotyped using whole genome amplification (WGA) followed by AmplideX® FMR1 PCR* (Asuragen). One, two or five cells were placed in PCR tubes under a dissecting microscope to monitor the release of the cell(s) into the tube; cell-line DNA was isolated using the DNeasy kit (Qiagen). Cell samples were tested in triplicates on three different days by two different laboratories (Cornell and Asuragen) and results were compared. Samples were also assessed using direct cell inputs into AmplideX FMR1 PCR* without upstream WGA.
Results: WGA of intact cells demonstrated superior sensitivity, repeatability, and reproducibility after FMR1 PCR compared to matched cell equivalents of purified gDNA. Using an optimized WGA-based protocol, both laboratories were successful in identifying normal and expanded genotypes from 1-5 cells in less than 48 hours. More specifically, FM alleles were accurately detected in 90% of replicate runs across the two sites when 5 cells were input into WGA. In addition, proof-of-concept was shown for the identification of expansions with up to 1200 repeats from single-cell FMR1 PCR—without WGA pre-amplification—in just 8 hours.
Figure 1. Summary of detection rates. FM alleles were accurately detected in 92% of replicate runs across the two sites when 5 cells were input into WGA. With 1 and 2 cell inputs into WGA, FM alleles were detected in at least 1/3 replicates, and in 5 cells at least 2/3 replicates were detected.
Conclusions: A novel fragile X amplification workflow performed by two different laboratories reveals that a combination of WGA and AmplideX FMR1 PCR* can enable accurate genotyping of 1-5 fragile X cells. This result augurs PGD applications for FXS using D5/6 trophectoderm clinical biopsies. A further benefit of the approach is that it generates microgram quantities of WGA DNA that can be utilized for other IVF-related genetic tests. Finally, the study establishes feasibility for fragile X detection from a single cell by a direct PCR method using AmplideX FMR1 PCR*, thereby pushing the boundaries of direct PCR amplification from single cells.
*For Research Use Only. Not for use in diagnostics procedures.
Watch the video: Single-cell Fragile X Molecular Analysis and Implications for Pre-Implantation Genetic Diagnosis in a Clinical Research Study – presented at the 2016 ACMG Annual Clinical Genetics Meeting