Single Cell Analysis of Mutations in the <i>APC</i> Gene

Abstract

Introduction: Mutation analysis of inherited monogenic diseases is an important aspect of preimplantation genetic diagnosis. Familial adenomatous polyposis of the colon is an autosomal dominant inherited disorder caused by mutations in the tumor suppressor gene adenomatous polyposis coli (APC). A characteristic of this severe disease is the development of thousands of polyps in the colon which untreated evolve into malignant colon carcinomas. Here we present a novel approach for the establishment of mutation detection in the APC gene in single cells applicable for preimplantation genetic diagnosis. Methods: Single fixed lymphocytes were isolated via laser microdissection and transferred to reaction centers of planar chemically structured glass slides via a recently developed horizontal low-pressure single particle adsorbing transfer system (SPATS). A multiplex nested polymerase chain reaction protocol in a 1-μl reaction volume, followed by sequencing and fragment length analysis was applied in order to detect mutations in the APC gene. Results: Reliable isolation and transfer of single lymphocytes was demonstrated. High amplification efficiency and low allelic drop out (ADO) rates for polymorphic microsatellite markers and mutation specific amplification products of various mutations in the APC gene were obtained from fixed single cells. Conclusions: This novel nanotechnological downscaling approach enables a reliable validation of genetic testing using diploid single lymphocytes, and will open a wide range of single cell diagnostics.

Profile of a Serial Killer: Cellular and Molecular Approaches to Study Individual Cytotoxic T‐Cells following Therapeutic Vaccination

“…The chemically modified glass slide wells can be used as single reaction centers for cell growth and PCR all in one place [92] in which cells are stimulated, washed, and dried in situ, avoiding numerous handling steps and cellular stress [93]. This platform has already been used for the genetic analysis of circulating tumor cells in small-scale clinical trials [94] and is particularly advantageous for multiplex real-time PCR [95], robotic micromanipulations, and laser microdissection [96]. This method has a high sensitivity with a detection level threshold of 32 pg of purified DNA, allowing the minisequencing of mitochondrial DNA down to a single lymphocyte [97].…”

Surface Acoustic Wave Actuated Lab-on-Chip System for Single Cell Analysis

“…As feasibility study for our LOC by is for instance a single cell assay: We have chosen this application example as it addresses most relevant questions in molecular analysis, dealing with minimum target amount of DNA, to avoid background noise of unwanted DNA, contamination risks and detection limits. By using this droplet-in-droplet configuration, single DNA base pair deletion could be detected after single cell isolation and processing [46]. When only a minute amount of DNA is available, the simultaneous amplification of polymorphic marker sequences combined with the amplification of the mutated gene region is important in order to avoid misdiagnosis due to preferential amplification, allelic drop out or contamination.…”

“…For the analyzed 1bp deletion, the results of the sequencing analysis genotyping of the mutation (Figure 4a). All major results of the single cell analysis are for example summarized in [46].…”