Notes

Moore's Regulation revisited through Intel computer chip density.
Investigation of the genome of organisms is one of the major basics in molecular biology to understand the complex organization of cells. While genomic DNA can easily be isolated from tissues or cell cultures of plant, animal or human origin, DNA extraction from single cells is still challenging. Here, we describe three techniques for the amplification of genomic DNA of fixed single circulating tumor cells (CTC) isolated from blood of cancer patients. This amplification is aimed to increase DNA amounts from those of one cell to yields sufficient for different DNA analyses such as mutational analysis including next-generation sequencing, array-comparative genome hybridization (CGH), and quantitative measurement of gene amplifications. Molecular analysis of CTC as liquid biopsy can be used to identify therapeutic targets in personalized medicine directed, e.g. against human epidermal growth factor receptor 2 (HER2) or epidermal growth factor receptor (EGFR) and to stratify the patients to those therapies.Whole genome amplification is required to ensure the availability of sufficient material for copy number variation analysis of a genome deriving from an individual cell. Here, we describe the protocols we use for copy number variation analysis of non-fixed single cells by array-based approaches following single-cell isolation and whole genome amplification. We are focusing on two alternative protocols, an isothermal and a PCR-based whole genome amplification method, followed by either comparative genome hybridization (aCGH) or SNP array analysis, respectively.Ancient mitochondrial DNA has been used in a wide variety of paleontological and archeological studies, ranging from population dynamics of extinct species to patterns of domestication. Most of these studies have traditionally been based on the analysis of short fragments from the mitochondrial control region, analyzed using PCR coupled with Sanger sequencing. With the introduction of high-throughput sequencing, as well as new enrichment technologies, the recovery of full mitochondrial genomes (mitogenomes) from ancient specimens has become significantly less complicated. Here we present a protocol to build ancient extracts into Illumina high-throughput sequencing libraries, and subsequent Agilent array-based capture to enrich for the desired mitogenome. Both are based on previously published protocols, with the introduction of several improvements aimed to increase the recovery of short DNA fragments, while keeping the cost and effort requirements low. This protocol was designed for enrichment of mitochondrial DNA in ancient or other degraded samples. However, the protocols can be easily adapted for using for building libraries for shotgun-sequencing of whole genomes, or enrichment of other genomic regions.Whole genome amplification is an invaluable technique when working with DNA extracted from blood spots, as the DNA obtained from this source often is too limited for extensive genetic analysis. Two techniques that amplify the entire genome are common. Here, both are described with focus on the benefits and drawbacks of each system. However, in order to obtain the best possible WGA result the quality of input DNA extracted from the blood spot is essential, but also time consumption, flexibility in format and elution volume and price of the technology are factors influencing system choice. https://www.selleckchem.com/products/bay-1161909.html Here, three DNA extraction techniques are described and the above aspects are compared between the systems.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).This protocol describes the use of a 16plex PCR for the purpose assessing DNA quality after isothermal whole genome amplification (WGA). In short, DNA products, generated by amplification multiple displacement amplification, are forwarded to PCR targeting 15 short tandem repeats (STR) as well as amelogenin generating up to 32 different PCR products. After amplification, the PCR products are separated via capillary electrophoresis and analyzed based on the obtained DNA profiles. Isothermal WGA products of good DNA quality will result in DNA profiles with efficiencies of >90 % of the full DNA profile.This chapter describes a simple and inexpensive multiplex PCR-based method to assess the quality of whole genome amplification (WGA) products generated from heat-induced random fragmented DNA. A set of four primer pairs is used to amplify DNA sequences of WGA products in and downstream of GAPDH gene in yielding 100, 200, 300, and 400 bp fragments. PCR products are analyzed by agarose gel electrophoresis and the respective WGA quality is classified according to the number of obtained PCR bands. WGA products that yield three or four PCR bands are considered to be of high quality and yield good results when analyzed by means of array comparative genome hybridization (CGH).The here described method of isothermal whole genome amplification (iWGA) uses a Phi29 DNA polymerase-based kit (Illustra GenomiPhi V2 DNA Amplification Kit) that amplifies minute quantities of DNA by multiple strand displacement upon random hexamer primer binding. Starting from genomic DNA or single cells this amplification yields up to 5 μg of iWGA product with fragment lengths of 10 kb and longer. As this amplification lacks the need of fragmenting DNA, its products are well suited for many downstream applications (e.g. sequencing and DNA profiling). On the contrary, degraded DNA samples are not supported by the nature of the amplification and are not well suited.Whole genome amplification (WGA) is a widely used technique allowing multiplying picogram amounts of target DNA by several orders of magnitude. The technique described here is based on heat-induced random fragmentation yielding DNA strands mainly ranging from 0.1 to 1 kb in length. The fragmented DNA is then subjected to library generation by annealing of adaptor sequences to both ends of the DNA fragments. Using primers hybridizing to the adapter sequences, the DNA is amplified by thermal cycling. This amplification typically yields > 2 mg DNA from a single cell, is suited for amplifying DNA isolated from (partly) degraded samples [e.g. formalin-fixed paraffin-embedded (FFPE) material] and works well when used for array-comparative genome hybridization (array-CGH).A polymerase chain reaction (PCR) in water droplets with water-in-oil emulsion (emulsion PCR) facilitates parallel amplification of a single-molecule template. The amplified DNA can be immobilized onto microbeads bound to primer DNA. The product, termed a "bead library", has various applications such as next-generation sequencing (NGS) and the directed evolution of various functional biomolecules. Here, we describe a method for genomic library construction on microbeads using emulsion PCR.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)].Single cells are increasingly used to determine the heterogeneity of therapy targets in the genome during the course of a disease. The first challenge using single cells is to isolate these cells from the surrounding cells, especially when the targeted cells are rare. A number of techniques have been developed for this goal, each having specific limitations and possibilities. In this chapter, five of these techniques are discussed in the light of the isolation of circulating tumor cells (CTC) present at extremely low frequency in the blood of patients with metastatic cancer from the perspective of pre-enriched samples by means of CellSearch. The techniques described are micromanipulation, FACS, laser capture microdissection, DEPArray, and microfluidic solutions. All platforms are hampered with a low efficiency and differences in hands-on time and costs are the most important drivers for selection of the optimal platform.The increasing interest towards cellular heterogeneity within cell populations has pushed the development of new protocols to isolate and analyze single cells. PCR-based amplification techniques are widely used in this field. However, setting up an experiment and analyzing the results can sometimes be challenging. The aim of this chapter is to provide a general overview on single-cell PCR analysis focusing on the potential pitfalls and on the possible solutions to successfully perform the analysis.Whole genome amplification (WGA) is a widely used molecular technique that is becoming increasingly necessary in genetic research on a range of sample types including individual cells, fossilized remains and entire ecosystems. Multiple methods of WGA have been developed, each with specific strengths and weaknesses, but with a common defect in that each method distorts the initial template DNA during the course of amplification. The type, extent, and circumstance of the bias vary with the WGA method and particulars of the template DNA. In this review, we endeavor to discuss the types of bias introduced, the susceptibility of common WGA techniques to these bias types, and the interdependence between bias and characteristics of the template DNA. Finally, we attempt to illustrate some of the criteria specific to the analytical platform and research application that should be considered to enable combination of the appropriate WGA method, template DNA, sequencing platform, and intended use for optimal results.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.
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