ABSTRACT
Comprehensive genome-wide analyses of single cells represent an important tool for clinical applications, such as pre-implantation diagnostic and prenatal diagnosis, as well as for cancer research purpose. For the latter, studies of tumor heterogeneity, circulating tumor cells (CTCs), and disseminated cancer cells (DCCs) require the analysis of single-cell genomes. Here we describe a reliable and robust array-based comparative genomic hybridization (aCGH) protocol based on Ampli 1™ whole genome amplification that allows the detection of copy number alterations (CNAs) in single cancer cells as small as 100 kb.
Subject(s)
DNA Copy Number Variations , Neoplastic Cells, Circulating , Female , Pregnancy , Humans , Comparative Genomic Hybridization , Genome-Wide Association Study , Embryo ImplantationABSTRACT
Modern molecular biology relies on large amounts of high-quality genomic DNA. However, in a number of clinical or biological applications this requirement cannot be met, as starting material is either limited (e.g., preimplantation genetic diagnosis (PGD) or analysis of minimal residual cancer) or of insufficient quality (e.g., formalin-fixed paraffin-embedded tissue samples or forensics). As a consequence, in order to obtain sufficient amounts of material to analyze these demanding samples by state-of-the-art modern molecular assays, genomic DNA has to be amplified. This chapter summarizes available technologies for whole-genome amplification (WGA), bridging the last 25 years from the first developments to currently applied methods. We will especially elaborate on research application, as well as inherent advantages and limitations of various WGA technologies.
Subject(s)
Genome , Genomics/methods , Nucleic Acid Amplification Techniques , Animals , HumansABSTRACT
This chapter describes a single-cell whole genome amplification method (WGA) that has been originally published under the name "Single Cell Comparative Genomic Hybridization (SCOMP)" (Klein et al., Proc Natl Acad Sci U S A 96(8):4494-4499, 1999). The method has recently become available commercially under the name "Ampli1(™) WGA Kit." It is a PCR-based technique for whole genome amplification (WGA) allowing comprehensive and quite uniform amplification of DNA from low quantities of input DNA material, in particular single cells. The method is based on a ligation-mediated adaptor linker PCR approach. In contrast to other PCR-based WGA approaches, both the primer design and mechanism underlying the fragmentation of genome are nonrandom, enabling high priming efficiency and deterministic fragmentation of template DNA. This is particularly important for the design of (diagnostic) assays targeting specific loci. Here, we describe the WGA protocol for amplification of single-cell genomes designed to provide high-quality material in quantity sufficient for a number of locus-specific and genome-wide downstream assays [e.g., targeted Sanger sequencing, restriction fragment length polymorphism (RFLP), quantitative PCR (qPCR), and array comparative genomic hybridization (CGH)].
Subject(s)
Genome , Genomics/methods , Nucleic Acid Amplification Techniques , Single-Cell Analysis/methodsABSTRACT
Laser microdissection (LMD) and whole genome amplification (WGA) are valuable tools to isolate, purify, and genetically analyze cancer cells from tissue sections. In this chapter, we describe a workflow for microdissecting small regions of interest from cancer tissue, i.e. formalin-fixed paraffin-embedded (FFPE) and cryo-conserved specimens, and subsequent whole genome amplification by a deterministic WGA approach (Ampli1™ WGA).
