Roberto Cruz-Flores, Jorge Cáceres-Martínez, Arun K Dhar
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引用次数: 0
Abstract
Laser microdissection (LMD) and whole genome amplification (WGA) are powerful techniques that integrate molecular and histological approaches to enable the precise selection of a minimal number of virus-infected cells-down to near single-cell resolution-and the subsequent generation of whole viral genomes with minimal host DNA interference. This chapter presents a detailed protocol for LMD and near single-cell WGA, specifically optimized for the recovery and sequencing of viral genomes from formalin-fixed paraffin-embedded (FFPE) tissues. The method allows for the targeted isolation of infected cells, thereby reducing host genomic background and enhancing the detection of pathogen-specific signals for downstream next-generation sequencing (NGS). The protocol includes steps for tissue section preparation, cell isolation via LMD, DNA extraction using the PicoPure DNA Extraction Kit, and unbiased genome amplification using the SeqPlex DNA Amplification Kit-ensuring high-quality nucleic acid recovery suitable for NGS workflows.
期刊介绍：
For over 20 years, biological scientists have come to rely on the research protocols and methodologies in the critically acclaimed Methods in Molecular Biology series. The series was the first to introduce the step-by-step protocols approach that has become the standard in all biomedical protocol publishing. Each protocol is provided in readily-reproducible step-by-step fashion, opening with an introductory overview, a list of the materials and reagents needed to complete the experiment, and followed by a detailed procedure that is supported with a helpful notes section offering tips and tricks of the trade as well as troubleshooting advice.
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