Rohan_version_MKBS_314-_study_unit_2.2_and_2.3

Study Unit Overview

• Title: MKBS 314 - Study Unit 2.3-4

• Instructor: Professor Rasheed Adeleke

• Subject: Microbiology

• Focus: Recombinant DNA Technology and Industrial Microbiology

Learning Outcomes

• Understanding PCR as a tool linking DNA applications and replication

• Discuss quality and quantity of PCR products and their analyses

• Explain principles and applications of real-time PCR (QPCR)

• Basic principles of reverse transcription and gene expression

Polymerase Chain Reaction (PCR) Overview

• PCR is a method used for amplifying DNA.

• It is crucial for various applications in molecular biology including genetic testing, research, and diagnostics.

Real-Time PCR (qPCR)

• Definition: A method that allows quantifying DNA during the PCR process, displaying the amplification in real time.

• Detection: Conducted during each cycle of the PCR product accumulation; tracks during early and exponential phases.

• Components: Includes dyes like SYBR Green, which binds to double-stranded DNA, and requires a laser for detection.

• Graph Representation: X-axis displays PCR cycles, and Y-axis shows logarithmic intensity; results are visualized in real-time.

Applications of Real-Time PCR

• Gene Expression Profiling

• miRNA Expression Analysis

• Pathogen Detection

• Viral Quantification

• Genetic Mutations and SNP Genotyping

Quantification Methods in PCR

Two Main Approaches:

1. Absolute Quantification

   • Develop a standard curve with known concentrations of target sequences.

   • Compare unknown sample's Ct value to generate concentration estimates.

2. Relative Quantification

   • Expression analyzes a target gene against a reference gene.

   • Normalizes variations, making use of the delta-delta Ct method to report fold changes.

Reverse Transcription PCR (RT-PCR)

• RT-qPCR: Combines reverse transcription and quantitative PCR.

• mRNA as Starting Material: Transcribed into complementary DNA (cDNA), then quantified.

Applications of qPCR

• Viral Pathogen Detection: Identifying specific viral particles such as HIV DNA copies.

• Genetic Disorders Detection: Screening for mutations linked to diseases like cancer.

• Forensics: Analyzing genetic material in criminal investigations and archaeological contexts.

Challenges with Pathogens in Water

• Non-Culturable Pathogens: Difficulty in directly culturing organisms present in water like Legionella pneumophila and Giardia.

• Monitoring Techniques: Concentrate organisms by filtration, isolating DNA and utilizing specific primers for detection.

PCR Comparison: qPCR vs. Conventional PCR

Key Importance of PCR in Biology

• Molecular Diagnostics: Essential for disease detection, including infectious diseases and genetic disorders.

• Genetic Research: Vital for gene cloning, mutation analysis, and epigenetic studies.

• Biotechnology: Plays a role in recombinant DNA technology and CRISPR gene editing implementations.

• Agriculture & Food Safety: Utilized for GMO detection and ensuring food safety through pathogen detection.

• Cancer Research: Involves tumor mutation analysis and guides personalized medicine through biomarker detection.

Gene and Restriction Enzymes

• Gene Structure: The functional unit of genetics consisting of nucleotide sequences coding for proteins.

• Restriction Enzymes: Recognize specific DNA sequences, cleaving strands, producing sticky or blunt ends, with hundreds available commercially.

Genetic Libraries

• Definition: Libraries are collections of cloned DNA, utilizing vectors and host cells for storage.

Types of Libraries:

• Genomic Library: Contains coding and non-coding sequences.

• cDNA Library: Contains only sequences of expressed genes.

Differences

• Genomic libraries include various sequences from the entire genome while cDNA libraries focus on expressed genes, not containing introns.

Reverse Transcription

• Discovery: Reverse transcriptase enzymes allow the conversion of RNA to DNA, enabling the analysis of mRNA.

• Process: Involves several steps from adding primers to synthesize cDNA, followed by cutting and generating a double-stranded DNA.