Week 13

Introduction

• Title: Molecular Techniques in Agriculture

• Author: Magida Tabbara, PhD Candidate

• Course: AGRI 3000 - Agricultural Genetics

• Date: Fall 2024, November 11th, 2024

Molecular Biology – Applications

• Molecular biology helps to:

  • Understand DNA's role in genetic information storage and protein expression.

  • Provide mechanisms for DNA manipulation by humans for various purposes.

Molecular Biology – Applications

• Applications categorized by objectives:

  1. Characterization: Identify individuals or populations through translation products (proteins) or directly via DNA.

  2. Genetic Alteration: Modify genetic information in an individual.

  3. Creation of New Combinations: Utilize recombinant DNA techniques to create genetic combinations not found in nature.

Aquaculture

• Focus: Salmon culture

Salmon Industry – Some Numbers

Production:

• ~70% of world salmon is farmed.

• 2022 figures:

• 2.8 million tons of farmed salmonids

The Necessity for Farmed Salmon

• Industry Value: $3 billion

• Nutritional benefits

• Market Size: 5.5kg (~12 lbs)

• Time to reach market size: 28 – 36 months.

Main Producer Countries of Salmon

• Countries include: Norway, Iceland, Finland, Faeroe Islands, UK, Russian Federation, Canada, Ireland, Denmark, France, USA, Spain, Greece, Turkey, Australia, Chile.

Potential Problems with Farmed Salmon

• Concerns include:

  • Escapees: Compete with wild juveniles for resources.

  • Diseases: Infections like ISA, SSSV, and issues with parasites affecting wild populations.

  • Environmental Impact: Waste from aquaculture operations harming ecosystems and wild fisheries.

A Molecular Solution

• Gene Editing Technologies: Change an organism's DNA without new DNA insertion

• CRISPR-Cas9: Most common system, consisting of a guide RNA that targets specific DNA sequences and the Cas9 enzyme that cuts the DNA, allowing for modifications

• AquAdvantage® Salmon: A genetically engineered Atlantic salmon developed to grow faster and reduce threats to wild populations by being sterile (triploid), minimizing competition for resources.

Altering Genetic Information without New DNA Insertion

• Gene Editing Technologies: Designed to change an organism's DNA.

  • Methods include: Removal of nucleotides, addition of nucleotides, or insertion of DNA segments.

  • CRISPR-Cas9: Most common gene editing system.

CRISPR Fundamentals

• CRISPR: Clustered Regularly Interspaced Short Palindromic Repeats

• Cas9: Enzyme with endonuclease activity, crucial for gene editing.

CRISPR-Cas9 Mechanism

• Involves a guide RNA targeting specific DNA sequences.

• Cas9 enzyme cuts the target DNA sequence recognizing a PAM (protospacer adjacent motif) sequence.

• Desired sequences can be added to repair DNA during this process.

CRISPR-Cas9 in Aquaculture

• CRISPR-Cas9 addresses sustainability challenges in aquaculture, such as knocking out the dnd gene in salmon.

Economic Considerations

• Farmers must also benefit from the adoption of genetic modifications.

AquAdvantage® Salmon

• Genetically engineered Atlantic salmon by AquaBounty Technologies, developed since 1989.

• Features growth hormone gene from Chinook salmon manipulated with a promoter from ocean pout.

Key Species Involved

• Salmon Species:

  • Atlantic salmon (Salmo salar)

  • Ocean pout (Zoarces americanus)

  • Chinook salmon (Oncorhynchus tshawytscha)

AquAdvantage® Salmon Benefits

• Market Size: 16 – 20 months compared to 28 – 36 months for traditional salmon.

• Efficient feed conversion: eats 25% less, 20% better feed efficiency.

• Continuous transgene expression promotes year-round growth.

Sterility of AquAdvantage® Salmon

• Reproductive Status:

  • Wild salmon: diploid (two sets of chromosomes)

  • AquAdvantage® Salmon: triploid (three sets, sterile).

  • This trait reduces threats to wild salmon populations.

FDA Approval of AquAdvantage Salmon

• FDA granted approval for AquAdvantage Salmon as a new animal drug in November 2015.

• Related documents are made available by the FDA.

Opinion on Aquaculture and Politics

• Discussion on North American aquaculture, environmental and regulatory issues surrounding salmon farming.

GMOs, Bioethics, and Biosecurity

GMOs are classified into three generations: 1st Generation includes crops for agronomic management (e.g., BT crops), 2nd Generation enhances consumer

DNA Sequencing Introduction

• Nucleotides: dNTPs (hydroxyl group for elongation) vs. ddNTPs (no hydroxyl, causes termination).

• Applications: Used in disease diagnostics, pathogen identification, and mutation analysis.

DNA Sequencing Overview

• Definition: Process of determining nucleotide order in DNA.

• Sanger Method:

  • Developed by Fred Sanger; Nobel Prize 1980.

  • Requires radioactive primers, DNA polymerase, and modified nucleotides (ddNTPs).

Steps in Sanger Sequencing

1. Denaturation: Split double-stranded DNA.

2. Annealing: Attach oligonucleotide primers.

3. Extension: DNA polymerase extends the DNA.

4. Termination: ddNTPs cause sequence termination.

5. Separation: DNA fragments sorted by size.

6. Detection: Read fragment order.

7. Sequence: Determine final sequence.

Comparison of dNTP and ddNTP

• Nucleotide Structure:

  • dNTP contains hydroxyl group, enabling further elongation.

  • ddNTP lacks hydroxyl group, leads to chain termination during sequencing.

Automated DNA Sequencing

• Utilizes capillary electrophoresis and fluorescence detection from ddNTPs.

Chain-Termination PCR Method

• The chain-termination polymerase chain reaction (PCR) method is pivotal in modern DNA sequencing techniques. This process specifically focuses on the size-separation and analysis of DNA fragments using fluorescently labeled dideoxynucleotides (ddNTPs) and capillary gel electrophoresis.

Chain-Termination PCR Method Summary

1. Template Preparation: Denature double-stranded DNA into single strands for primer access.

2. Primer Annealing: Primers bind to the template, initiating DNA synthesis.

3. Extension: DNA polymerase extends primers with standard (dNTPs) and fluorescent ddNTPs.

4. Termination: Incorporation of ddNTPs halts synthesis, creating truncated DNA fragments.

5. Separation: DNA fragments are sorted by size using capillary electrophoresis, with smaller fragments moving faster.

6. Detection and Analysis: Fluorescent signals from ddNTPs are recorded, producing a chromatogram that indicates the nucleotide order. This high-throughput method is vital for genomics and diagnostics.

This method allows for high-throughput and efficient sequencing, crucial for applications in genomics, medical diagnostics, and biological research.

Applications

• DNA sequencing applicable in disease diagnostics, pathogen identification, and mutation analysis.

Page 32: Alteration of Hereditary Material

• Gene editing without DNA insertion utilizes CRISPR-Cas9 to remove or add nucleotides or DNA segments. This mechanism, derived from bacteria's viral defense, uses RNA to guide the Cas9 enzyme for DNA cutting. Essential requirements for CRISPR include PAM sites in DNA, matching gRNA, active Cas9, and a delivery system for components into target cells.

Gene Editing without DNA Insertion

• CRISPR-Cas9 Mechanism: Achieved by removal/addition of nucleotides or DNA segments.

CRISPR-Cas9 in Nature

• Adapted from bacterial defense against viruses, utilizing CRISPR arrays for recognition.

CRISPR Mechanism

• Bacteria produce RNA (gRNA) that guides Cas9 to cut virus DNA, effectively disabling it.

Steps in Gene Editing with CRISPR-Cas9

1. Recognition: gRNA directs Cas9 to specific DNA.

2. Cleavage: Cas9 cuts double-stranded DNA.

3. Repair: DNA repair processes fix double helix based on the presence of donor DNA.

CRISPR Requirements

1. PAM site in target DNA.

2. gRNA matching the genomic sequence.

3. Active Cas9 protein complex.

4. Delivery system for components into target cells.

5. Selection method for transformed individuals.

Recombinant DNA Techniques

• Definition: Manipulation and isolation of DNA segments using enzymes.

• Transgenics: Insertion of foreign DNA into an organism’s genome, distinguishing it from broader GMO classification.

Steps in Gene Cloning

1. Isolate DNA segment.

2. Insert into vector to create recombinant DNA.

3. Introduce recombinant DNA into host.

4. Select transformed cells with gene of interest.

5. Multiply and express gene in host.

6. Isolate and purify gene copies.

Cloning Vectors

• Types include plasmids, bacteriophages, and artificial chromosomes (BACs, YACs, MACs).

• Plasmids and bacteriophages are the most commonly used types.

Gene Transfer Methods

• Plants: Techniques include Agrobacterium, particle bombardment.

• Animals: Microinjection with nuclear transfer for cloning; more complex than plant gene transfers.

Agrobacterium-Mediated Gene Transfer

• Natural process where Agrobacterium inserts Ti plasmid into plant cells, altered for gene insertion.

Agrobacterium Process Overview

• Process includes the creation of recombinant plasmids followed by gene insertion into plant cells using restriction enzymes and ligases.

Particle Bombardment Technique

• Describes how the bombardment chamber feeds gas and how DNA-coated particles are propelled into target cells.

Transformation Steps via Particle Bombardment

1. Mother plant cultivation

2. Isolation of leaf cells

3. Removal of cell walls to create protoplasts.

4. Gene transfer and callus induction leading to plantlet regeneration.

Microinjection Technique in Gene Transfer

• Involves using a glass pipette for inserting DNA directly into cell nucleus.

Types of Genetically Modified Organisms (GMOs)

• 1st Generation: Crops for agronomic management (e.g., BT crops).

• 2nd Generation: Crops for improved consumer quality (e.g., Golden Rice).

• 3rd Generation: Plants for the production of compounds such as biopolymers.

Genetically Modified Farm Animals

• Designed to produce drugs for human medical use (e.g., chickens and goats engineered for specific proteins).

• Goals include increased growth rates and enhanced productivity.

Bioethics and Biosafety Overview

• Bioethics: Moral principles guiding biological research and its impact on affected populations.

• Biosafety: Integrated approach to managing risks related to GMOs and their environmental impact.

Reading Summary

PCR-Based Techniques Using Specific Primers

• Adaptation in Primer Design

  • An additional nucleotide is added to the sequence of the adapter.

  • Primer anneals to ligated fragments containing this additional nucleotide.

Microsatellites (SSRs)

• Characteristics

  • High mutation rate leads to variability.

  • Regions with variable repetitions in their sequences.

• Primer Design

  • Requires knowledge of the species' genome.

  • Co-dominant; allows differentiation between homozygous (one band) and heterozygous individuals (two bands).

ISSR (Inter-Simple Sequence Repeat)

• Derived from Microsatellites

  • PCR with primers complementary to microsatellites amplifies fragments between these sites.

  • Typical primer length is between 16-25 bp.

Internal Transcribed Spacer (ITS)

• Overview

  • Sequences separate RNA transcripts of ribosomal RNA genes.

  • Prokaryotes: One ITS present; Eukaryotes: Two ITS.

  • Highly conserved flanking sequences allow primer design for comparison of DNA fingerprints.

DNA Sequencing

• Definition

  • Determining the nucleotide order in DNA segments.

  • DNA polymerase synthesizes new strands using a template strand with complementary nucleotides.

• Mechanism

  • 3′ hydroxyl group is essential for forming phosphodiester bonds between nucleotides.

Sanger’s DNA Sequencing Method

• Process

  • Uses dideoxynucleotides (chain termination) to halt DNA elongation.

  • Four separate PCR reactions for adenine, thymine, cytosine, and guanine.

• Output

  • Fragments of varying lengths are produced, determined by which dideoxynucleotide terminates the elongation.

  • Electrophoresis reveals DNA sequence through band visualization.

• Fluorescent Dyes

  • Different colors for each dideoxynucleotide improve detection.

  • Allows one-tube PCR with separation by size in capillary electrophoresis.

Molecular Marker-Assisted Selection

• Overview

  • Allows for selecting for traits based on specific DNA segments.

  • Takes advantage of linkage between certain DNA markers and phenotypic traits.

• Application Example

  • Evaluating beans for resistance to southern mosaic virus using zymograms.

• Advantages

  • Saves time in identifying desirable traits without phenotypic evaluation.

Genetic Editing vs. Recombinant DNA

• Genetic Editing

  • Techniques to manipulate genomes without inserting DNA directly (e.g., CRISPR-Cas9).

• Recombinant DNA

  • Allows gene transfer across species, creating genetically modified organisms (GMOs).

• Definition

  • GMOs are organisms with modified genomes through human intervention.

Gene Transfer Techniques

• Cloning and Isolation of Genes

  • Involves creating gene libraries using restriction enzymes to DNA digest.

  • Cloned genes are identified and isolated using complementary RNA probes.

Transformation Techniques

• Agrobacterium Method

  • Utilizes soil bacteria to transfer plasmids into plant cells.

• Biolistics

  • Gene gun method using microparticles to drive DNA into cells.

Applications of GMOs in Agriculture

• Types

  • First-generation: Crops altered for easier farming (e.g., BT corn).

  • Second-generation: Crops designed for higher consumer quality (e.g., golden rice).

  • Third-generation: Crops as biological factories for compounds.

Bioethics and Biosafety Concerns

• Bioethics

  • Moral principles governing biological research and its impact on life.

• Biosafety

  • Evaluating risks related to GMOs for humans, animals, plants, and the environment.

• Importance

  • Ensuring GMOs do not pose greater risks than conventional alternatives.