Molecular Microbiology: DNA/RNA Extraction, PCR, Real-Time PCR, and Sequencing Applications

https://doi.org/10.66715/cerebral-link/2026.v4.c12.126142 | Cerebral Link | Publish by Cerebral Publication Private Limited | 2026 | Volume 4 | Page 126-142 |ISBN: 978-81-689463-6-1 | Book Title: - Text-book of Practical Microbiology | Chapter 12: Molecular Microbiology: DNA/RNA Extraction, PCR, Real-Time PCR, and Sequencing Applications

Author:- Dr Onkar Nath Tiwari, Assistant Professor, Department of Biochemistry, Shri Gorakhnath Medical College Hospital and Research Centre, UP, India

Molecular Microbiology: DNA/RNA Extraction, PCR, Real-Time PCR, and Sequencing Application.

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Abstract

Molecular microbiology has transformed the detection, identification, characterization, and surveillance of microorganisms by enabling highly sensitive and specific analysis of microbial nucleic acids. This chapter provides a comprehensive overview of essential molecular techniques, including DNA and RNA extraction, polymerase chain reaction (PCR), real-time PCR (qPCR), and sequencing technologies. It discusses the fundamental principles of nucleic acid isolation, sample preparation, quality assessment, primer design, amplification, and interpretation of molecular results. Conventional PCR and real-time PCR are examined with emphasis on their applications in rapid pathogen detection, microbial quantification, gene expression analysis, antimicrobial resistance testing, and clinical diagnosis. The chapter also introduces Sanger sequencing and next-generation sequencing (NGS), highlighting their roles in microbial identification, whole-genome analysis, outbreak investigation, metagenomics, and epidemiological surveillance. Key considerations such as contamination control, quality assurance, biosafety, analytical sensitivity, specificity, and troubleshooting are addressed. Emerging advances in automation, multiplex molecular assays, portable sequencing, and bioinformatics are also discussed. Collectively, these molecular approaches provide powerful tools for modern microbiology, supporting accurate diagnosis, research, public health surveillance, and a deeper understanding of microbial diversity, evolution, and pathogenesis.