MEVE 018: Unit 11 – Biochemical and Molecular Techniques

UNIT 11: BIOCHEMICAL AND MOLECULAR TECHNIQUES

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11.0 Introduction

Biochemical and molecular techniques are fundamental tools in modern biology and environmental science. These techniques enable the analysis of DNA, RNA, proteins, and other biomolecules, helping researchers detect genetic variations, identify organisms, and monitor environmental contaminants at the molecular level. This unit introduces essential molecular tools like PCR, restriction enzymes, DNA fingerprinting, blotting, sequencing, and their applications in environmental monitoring.

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11.1 Objectives

After completing this unit, you should be able to:

• Understand the basic principles of restriction endonucleases and PCR.
• Explain DNA fingerprinting and its types (RFLP, RAPD, AFLP).
• Describe blotting techniques and sequencing methods.
• Apply these molecular techniques in environmental monitoring.

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11.2 Restriction Endonucleases

Restriction endonucleases, or restriction enzymes, are enzymes that recognize specific sequences of nucleotides in DNA and cut at or near these sites.

Types:

• Type I: Cleaves DNA at random far from the recognition site.
• Type II: Most commonly used; cuts within the recognition sequence.
• Type III: Cuts a short distance from the recognition site.

Applications:

• DNA cloning
• Gene mapping
• Preparing DNA for fingerprinting
• Environmental microbial identification

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11.3 Polymerase Chain Reaction (PCR)

PCR is a technique used to amplify a specific DNA segment exponentially.

Principle:

PCR involves repeated cycles of:

1. Denaturation (94–98°C): DNA strands are separated.
2. Annealing (50–65°C): Primers bind to target sequences.
3. Extension (72°C): DNA polymerase synthesizes new strands.

Applications:

• Detection of pathogens in water, air, and soil
• Monitoring genetically modified organisms (GMOs)
• Identification of endangered species through eDNA

Variants:

• RT-PCR (for RNA detection)
• qPCR (quantitative PCR)

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11.4 DNA Fingerprinting

DNA fingerprinting refers to analyzing DNA profiles to identify genetic differences among organisms.

11.4.1 RFLP (Restriction Fragment Length Polymorphism)

• Based on variations in DNA fragment lengths produced by restriction enzymes.
• Useful for comparing microbial strains in polluted environments.

11.4.2 RAPD (Random Amplified Polymorphic DNA)

• Amplifies random DNA segments using short primers.
• Rapid method, but low reproducibility.

11.4.3 AFLP (Amplified Fragment Length Polymorphism)

• Combines restriction digestion and selective PCR amplification.
• Highly sensitive for detecting genetic diversity in microbial communities.

Applications:

• Species identification
• Tracking genetic mutations due to pollution
• Studying microbial diversity in contaminated sites

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11.5 Blotting Techniques

Blotting involves transferring nucleic acids or proteins onto membranes for detection using labeled probes or antibodies.

Types:

• Southern Blot: DNA detection
• Northern Blot: RNA detection
• Western Blot: Protein detection

Process:

1. Gel electrophoresis
2. Transfer to membrane
3. Hybridization with probe (DNA/RNA/protein)
4. Detection via radioactive or chemiluminescent tags

Environmental Uses:

• Detecting gene expression in response to toxins
• Identifying pollutant-specific microbial genes

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11.6 Sequencing of Nucleic Acids and Proteins

Sequencing determines the exact order of nucleotides in DNA/RNA or amino acids in proteins.

DNA/RNA Sequencing:

• Sanger Sequencing: Chain termination method using labeled dideoxynucleotides.
• Next Generation Sequencing (NGS): High-throughput, parallel sequencing.

Protein Sequencing:

• Edman degradation: Sequential removal of amino acids from the N-terminus.
• Mass spectrometry: Identifies peptide masses and sequences.

Applications in Environment:

• Identifying microbial species in ecosystems
• Analyzing functional genes involved in bioremediation
• Understanding environmental metagenomes

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11.7 Applications in the Field of Environmental Monitoring

Molecular techniques are integral to environmental biotechnology and ecology.

Key Applications:

1. Pathogen Detection:
• PCR and qPCR for detecting E. coli, Salmonella, and viruses in water bodies.
2. GMO Monitoring:
• PCR and sequencing to detect genetically modified crops in the environment.
3. Bioremediation Tracking:
• Identifying microbes with genes for degrading pollutants (e.g., hydrocarbons, pesticides).
4. Biodiversity and Conservation:
• RAPD, AFLP, and sequencing for studying genetic diversity and endangered species.
5. Soil and Water Quality Analysis:
• Blotting techniques and molecular probes to assess functional gene expression.
6. Climate Change Studies:
• Monitoring microbial community shifts due to changing environmental parameters.

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11.8 Let Us Sum Up

This unit explored the fundamental biochemical and molecular biology tools used in environmental monitoring. Techniques such as PCR, restriction enzyme digestion, DNA fingerprinting (RFLP, RAPD, AFLP), blotting, and sequencing have become essential in assessing microbial activity, pollutant degradation, and biodiversity. These tools offer precise, rapid, and scalable methods to analyze complex environmental samples.

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11.9 Glossary

• PCR-Polymerase Chain Reaction; a method to amplify DNA.
• Restriction Endonucleases-Enzymes that cut DNA at specific sequences.
• RFLP-Technique for comparing DNA fragment lengths after restriction enzyme digestion.
• RAPD-A PCR-based technique using random primers to amplify genetic material.
• AFLP-A sensitive DNA fingerprinting technique combining digestion and selective PCR.
• Blotting-Transfer of macromolecules onto membranes for detection.
• Sequencing-Determination of nucleotide or amino acid order.
• NGS-Next Generation Sequencing; high-throughput DNA sequencing method.
• Metagenomics-Study of genetic material recovered directly from environmental samples.
• Bioremediation-Use of organisms to detoxify environmental contaminants.