Chapter 4

DNA Manipulation

• To work with DNA, having more DNA molecules is essential because having a larger sample amount allows more experiments to be performed.

• PCR (Polymerase Chain Reaction):

  • Used for DNA amplification.

  • Creates millions of identical DNA copies.

  • Enables analysis of trace DNA (e.g., from blood at a crime scene) or specific genes.

• Gel Electrophoresis:

  • Separates and visualizes nucleic acids and proteins based on size.

  • Often performed after PCR to:

    • Confirm successful DNA fragment amplification.

    • Identify DNA fragments in a sample (DNA profiling).

  • Also used to prepare DNA fragments for further manipulation.

DNA Amplification

• Need for DNA Amplification:

  • Many DNA techniques require large amounts of DNA.

  • Sometimes only small DNA samples are available (e.g., crime scenes, fossils, medical tests, embryonic/fetal screening).

  • Amplification increases the amount of DNA for use in other techniques.

• PCR and Target DNA:

  • PCR is used for DNA amplification.

  • PCR creates a large quantity of DNA identical to the initial small sample.

  • "Target DNA" refers to the specific DNA region (e.g., gene, microsatellite) being studied or manipulated.

Polymerase

• PCR and Polymerases:

  • PCR is based on the action of polymerases.

  • Polymerases are enzymes that create long-chain molecules (polymers) like DNA and RNA by linking nucleotides.

  • These enzymes are found in all living organisms

  • Polymerases are essential for DNA replication, repair, and maintenance.

• Types of Polymerases:

  • DNA polymerases

  • RNA polymerases

DNA polymerase

• DNA Polymerase in PCR and Sequencing:

  • Used to synthesize multiple copies of target DNA.

  • Attaches to the template DNA and adds complementary nucleotides, creating a new DNA strand.

• Requirements for PCR DNA Polymerases:

  • Stable at high temperatures.

  • High affinity for the DNA template.

  • High specificity to minimize amplification of non-target DNA.

Taq Polymerase

• Most common DNA polymerase used in PCR.

• Originally extracted from the thermophilic bacterium Thermus aquaticus.(BT)

• Heat-resistant properties make it ideal for PCR and other DNA manipulation techniques.

Reverse Transcriptase

• Reverse Transcriptase:

  • A DNA polymerase that synthesizes single-stranded DNA using single-stranded RNA as a template.

  • Reverses the usual transcription process (DNA to RNA).

• Uses of Reverse Transcriptase:

  • Produces DNA molecules that can be amplified by PCR.

  • Creates complementary DNA (cDNA) from modified mRNA (introns removed) for some DNA manipulation techniques.

RNA polymerase

• RNA Polymerases:

  • Synthesize RNA from DNA during transcription.

  • Subunits recognise the promoter at the start of a gene.

  • Attaches to the promoter, unwinds DNA, and adds nucleotides (5' to 3' direction).

  • Continues until a "stop" sequence is reached, creating a single-stranded RNA molecule.

  • Works slower than DNA polymerase.

• Uses of RNA Polymerase:

  • Used in specialized lab techniques to study transcription and RNA amplification.

Polymerase Chain Reaction

How PCR works:

• Cyclic Process: PCR involves repeated cycles of heating and cooling, each cycle doubling the amount of target DNA. This exponential amplification leads to a massive increase in the number of DNA copies.

• Exponential Growth: Starting with just a few molecules of DNA, after 30 cycles, PCR can generate over a billion copies of the target DNA sequence.

• PCR Mixture: For PCR to occur, a specific mixture is required:

  • DNA: This includes the target DNA sequence to be amplified.

  • Free Nucleotides: These are the building blocks of DNA, necessary for synthesizing new DNA strands.

  • DNA Polymerase: A heat-resistant enzyme, such as Taq polymerase, that can withstand the high temperatures used in PCR. This enzyme helps in the elongation of new DNA strands by adding free nucleotides.

  • DNA Primers: Two short, single-stranded DNA molecules, typically up to 30 bases long. These primers are designed to be complementary to the ends of the target DNA sequence, ensuring that only the desired segment is amplified. They specify the start and finish of the DNA fragment to be amplified.

• Specialized Equipment: PCR requires a specialized machine called a thermal cycler, which can precisely control the temperature changes needed for each cycle.

• PCR Overview:

  • Method for amplifying specific target DNA sequences.

  • Uses Taq polymerase (heat-stable DNA polymerase).

  • Each DNA strand acts as a template for a new copy.

• PCR Cycles:

  • Each cycle doubles the DNA amount (exponential growth).

  • 30 cycles result in over one billion copies.

• PCR Requirements:

  • Specialized equipment.

  • PCR mixture containing:

    • DNA sample (including target DNA).

    • Free nucleotides.

    • Taq polymerase.

    • Two DNA primers (complementary to target DNA ends, specifying start and finish of amplification).

• Process

  1. A DNA sample including the target DNA to be amplified

  2. A heat resistant DNA polymerase (Taq polymerase) used to elongate the new DNA strands by adding free nucleotides

  3. Free nucleotides to build the new strand which is complementary to single-stranded DNA

  4. Two DNA primers complementary to ends of target DNA to specify start and finish of DNA fragment to be amplified

• PCR Process: The PCR mixture is placed in a thermocycler, which controls temperature changes. Each cycle has three steps:

  1. Denaturation (95°C): Heat breaks hydrogen bonds, separating double-stranded DNA into single strands.

  2. Annealing (50-60°C): Primers bind to complementary sequences on the single-stranded DNA at the target sequence ends.

  3. Elongation (72°C): Taq polymerase attaches to the primers and adds nucleotides, creating new complementary DNA strands.

• Cycle Repetition: This three-step cycle is repeated up to 50 times to produce a large amount of target DNA.

DNA Seperation

• Gel Electrophoresis and PCR: Gel electrophoresis is often used after PCR.

• Purpose of Gel Electrophoresis:

  • Separates DNA fragments based on size.

  • Used for:

    • DNA profiling (comparing fragments from different samples).

    • Isolating specific fragments for other techniques (e.g., DNA recombination, bacterial transformation).

Gel Electrophoresis

• Gel Electrophoresis Overview:

  • Technique for separating nucleic acid (DNA, RNA) fragments and studying proteins (focus here is on DNA).

  • Negatively charged DNA fragments move through the gel towards the positive terminal when an electric current is applied.

  • Smaller fragments move faster than larger fragments, separating them by size.

• Uses of Gel Electrophoresis:

  • DNA screening (e.g., genetic conditions).

  • Confirming correct gene amplification in PCR.

  • Identifying DNA fragments for genetic engineering.

• Gel Electrophoresis Process:

  1. Gel Preparation: Agarose gel (jelly-like) with wells at one end.

  2. Gel Placement: Gel placed in electrophoresis chamber with wells at the negative terminal.

  3. Sample Loading: DNA samples loaded into the wells.

  4. DNA Ladder: A DNA ladder (fragments of known lengths) is also run for comparison and size estimation of sample fragments.

  5. Electrophoresis Bath: Gel placed in a bath with a controlled pH solution containing ions for conductivity.

  6. Power Application: Electric current applied, causing DNA fragments to migrate towards the positive terminal.

  7. Fragment Seperations: Smaller fragments move faster through the gel, so they migrate further through the gel than larger fragments. This sorts the fragments by length.

  8. Visualisation: The DNA fragments are made visible by applying a stain that binds to DNA. This can be done with a fluorescent stain.

     • (which may be included in the gel or added after)

     • Staining (fluorescent or methylene blue).

     • Fluorescent: Viewed under UV light.

     • Methylene blue: Visible to the eye.

     • Bright bands indicate DNA presence.

Combining Molecular tools to detect Mutations

• Mutations are often identified by their effects on the individual.

• Cystic fibrosis (CF) symptoms can prompt analysis of the CFTR gene to detect mutations.

• The CFTR gene codes for a membrane protein that regulates chloride ion movement.

• Many different mutations in the large CFTR gene can cause CF.

• The most common mutation, ΔF508, is a deletion of three base pairs, removing phenylalanine at position 508.

• Families with CF history may be screened for this and other common CF mutations.

• PCR and gel electrophoresis can detect the ΔF508 mutant allele:

  • DNA is isolated (mouth swab or amniotic fluid).

  • PCR primers amplify the region surrounding the ΔF508 mutation site.

  • Amplified DNA is compared using gel electrophoresis.

  • Normal allele: 98 base pairs.

  • ΔF508 mutant allele: 95 base pairs.

  • A DNA ladder helps identify normal and mutant alleles based on size.

DNA Profiling

• DNA profiling identifies individuals based on unique DNA sequences.

• Applications include forensics, disaster victim identification, and paternity testing.

• It relies on variations in non-coding DNA (introns) called polymorphisms.

• Short tandem repeats (STRs) or microsatellites are used for identification.

• STRs are short, repeated DNA sequences (2-6 bases).

• Thousands of STR loci exist, and ~20 are typically used in profiling.

• Homologous chromosomes have the same STR sequence, but the number of repeats can differ between individuals.

• This difference in repeat number at various STR loci creates a unique DNA profile for each person.

Techniques involved:

• DNA profiling is sensitive and can use small or degraded samples (blood, semen, saliva, hair).

• Process:

  • DNA extraction.

  • DNA digestion with restriction enzymes.

  • STR amplification using PCR with specific primers for each.

  • STR size detection via gel or capillary electrophoresis.

• Capillary electrophoresis:

  • DNA fragments move through a thin tube under an electric field.

  • Smaller fragments move faster.

  • A laser detector registers peaks on a graph.

  • Two copies of each STR (one per chromosome) usually result in two peaks.

  • Identical STR copies result in a single peak.

• A DNA profile is generated from STR analysis.

• Crime scene DNA is compared to suspect DNA.

• A match occurs when STR lengths at all tested sites are identical between the crime scene sample and the suspect's DNA.

• A perfect match across 20 STRs indicates an extremely low probability (hundreds of billions to one) that the samples are from different people.

• DNA profiling is now being used to predict physical appearance (eye, skin, hair color) and sometimes ancestry.

• This aspect of DNA profiling is still developing and may not be completely reliable yet.

Bacterial Transformation

• Plasmids are small, circular DNA molecules in bacteria, often used as vectors.

• Genes can be inserted into plasmids, then introduced into bacteria via transformation.

• The plasmid replicates, also replicating the inserted gene.

• The bacteria express the inserted gene, producing the corresponding protein (e.g., insulin).

• Restriction enzymes and ligases are used to create recombinant DNA, using plasmids as vectors.

Restriction Enzymes (Endonucleases)

• DNA molecules are large, making them difficult to work with.

• Restriction enzymes (endonucleases) cut DNA into smaller, manageable fragments.

• They are naturally found in bacteria as a defense against foreign DNA (e.g., bacteriophages).

• Bacteria protect their own DNA from restriction enzymes via methylation.

• Each restriction enzyme recognizes and cuts a specific 4-6 base pair sequence (recognition site).

• Cutting occurs on the phosphodiester backbone of each DNA strand.

• Two types of restriction enzymes:

  • Sticky-end enzymes:

    • Cut DNA at different locations on each strand within the recognition site, creating staggered cuts and "sticky ends" (overhanging bases).

    • Sticky ends can hydrogen bond with complementary sticky ends.

    • Example: EcoRI (from E. coli) recognizes GAATTC (a palindromic sequence) and cuts between G and A.

  • Blunt-end enzymes:

    • enzymes cut both DNA strands at the same location within the recognition site.

    • This results in "blunt" or clean-cut ends, with no overhanging bases.

    • HaeIII (from Haemophilus aegyptius) is an example.

    • HaeIII recognizes GGCC and cuts between the G and C, creating blunt ends.

Identifying Polymorphisims and Mutations

• Polymorphisms are small DNA sequence variations within a population.

• A variation must have a frequency of 1% or more to be considered a polymorphism, not a mutation.

• Mutations can create new allele variants.

• Example: Sickle-cell anemia is caused by a mutation in the β-globin gene.

• This mutation is a missense mutation: a single base change (A to T) leading to an amino acid substitution in the β-globin protein.

• The altered β-globin causes hemoglobin to clump, reducing oxygen capacity and distorting red blood cells into a sickle shape, leading to various health problems.

• Sickle Cell Mutation: The mutation causing sickle-shaped red blood cells happens within a restriction enzyme recognition site.

• MstII Enzyme: This specific mutation eliminates the recognition site for the MstII restriction enzyme.

• Detection Method: Uses PCR, restriction enzyme digestion, and gel electrophoresis.

• DNA Extraction: DNA is extracted from the individual.

• PCR Amplification: The DNA region containing the MstII site is amplified using PCR.

• Restriction Digestion: The PCR product is incubated with MstII.

  • Normal Allele: MstII cuts the DNA at its recognition site (two fragments).

  • Mutant Allele: MstII cannot cut the DNA (one fragment).

• Gel Electrophoresis: DNA fragments are separated by size.

  • Normal Allele: Two bands on the gel.

  • Mutant Allele: One band on the gel.

• Result: The banding pattern on the gel reveals whether an individual carries the normal, mutant, or both alleles.

Ligase

• Ligases: Enzymes that join DNA or RNA fragments.

• Two Types:

  • DNA Ligases: Join DNA fragments.

  • RNA Ligases: Join RNA fragments.

• Cellular Role of DNA Ligase:

  • Joins newly replicated DNA segments.

  • Repairs breaks in DNA molecules.

• Joining DNA from Different Sources: DNA ligase can join DNA fragments from different organisms or species because DNA's molecular structure is universally consistent. This is a key principle in recombinant DNA technology.

• Laboratory Conditions: For efficient ligation in the lab, conditions must be optimized based on:

  • DNA ends: "Sticky ends" (overhangs) ligate more easily than "blunt ends."

  • Reaction conditions: Incubation time and temperature are crucial for optimal enzyme activity.

Ligation of DNA Fragments:

• Sticky Ends:

  • Specific and efficient.

  • Complementary base pairing between overhangs (sticky ends) provides initial binding.

  • DNA ligase creates phosphodiester bonds, joining the fragments.

  • Used for recombinant DNA and gene cloning.

• Blunt Ends:

  • Random and less efficient.

  • Any two blunt ends can join.

  • More difficult for specific fragment joining.

  • Sometimes unavoidable (e.g., when a specific restriction enzyme is needed).

  • Can be modified by adding "linker" DNA fragments to create sticky ends.

Recombinant DNA:

• Definition: DNA from two different species joined together.

• Purpose: To clone (make many copies of) a specific gene and/or produce the protein encoded by that gene.

• Example: Cloning the insulin gene into bacteria to produce human insulin.

Insulin Production:

• Previous Method: Insulin was extracted from animal pancreases (pigs, cattle). This was expensive, time-consuming, and carried risks of allergic reactions and disease transmission. Also, animal insulin is not identical to human insulin and less effective.

• Modern Method: Recombinant DNA technology is used to produce human insulin in bacteria. This is more efficient, safer, and produces human insulin, which is more effective. Recombinant human insulin became available in the 1980s.

Plasmid as Vectors

• Process:

  1. Target DNA Insertion: The gene of interest (e.g., the INS gene for insulin) is inserted into a plasmid, creating a recombinant plasmid.

  2. Transformation: The recombinant plasmid is introduced into a bacterial cell.

  3. Replication and Expression: The plasmid replicates within the bacteria, and the bacteria express the genes on the plasmid, including the inserted gene (insulin).

Advantages of Using Plasmids:

• Small Size: Easy to manipulate in the lab.

• Multiple Restriction Sites: Plasmids are engineered to contain various restriction enzyme sites, allowing flexibility in choosing enzymes for inserting the target gene. The restriction site in the plasmid must match the restriction site used to cut the gene of interest.

• Self-Replication: Plasmids replicate independently and faster than the bacterial chromosome, leading to high copy numbers of the gene of interest and thus, high levels of protein production.

Essential Plasmid Characteristics:

• Antibiotic Resistance Gene: Allows for selection of bacteria that have taken up the plasmid. Only bacteria containing the plasmid (and thus the antibiotic resistance gene) will survive in the presence of the antibiotic.

• Reporter Gene: A gene that produces a visible or easily identifiable product (e.g., a colored or fluorescent protein). This helps confirm that the plasmid has been taken up and, in some cases, whether the target gene has been successfully inserted.

Example: lacZ Gene as a Reporter:

• The lacZ gene, which encodes an enzyme that can break down a specific sugar, is often used as a reporter gene.

• Restriction sites for gene insertion are located within the lacZ gene.

• If the gene insertion is successful, the lacZ gene is disrupted and can no longer produce its enzyme. This disruption is used to identify bacteria containing the recombinant plasmid.

• The plasmid also contains the ampR gene, conferring ampicillin resistance.

Recombinant DNA Creation

1. INS Gene Isolation: The insulin gene (INS) is cut out of its source DNA using a sticky-end restriction enzyme and isolated.

2. Plasmid Preparation: A bacterial plasmid is cut with the same restriction enzyme, creating complementary sticky ends on the plasmid.

3. Combining DNA: The INS gene and plasmid are mixed. Some plasmids rejoin without the INS gene (non-recombinant), while others incorporate the INS gene (recombinant). Reporter genes are crucial for distinguishing between these.

4. Ligation: DNA ligase is added to seal the DNA backbones, creating stable recombinant plasmids.

Complimentry DNA

• Eukaryotic genes contain introns: Eukaryotic genes have non-coding regions called introns that interrupt the protein-coding regions (exons). Bacteria cannot process introns.

• mRNA processing: In eukaryotes, introns are removed from the initial RNA transcript to produce mature mRNA, which only contains exons.

• cDNA as a copy of mature mRNA: cDNA is created by using reverse transcriptase to make a DNA copy of mature mRNA. This cDNA therefore lacks introns.

• Bacterial compatibility: Because cDNA only contains the protein-coding exons, it can be correctly transcribed and translated into protein by the bacterial cell.

Reverse Transcriptase

• This is useful because mature mRNA has already had the introns spliced out. Prokaryotic cells are unable to splice out introns.

• Reverse transcriptase allows the synthesis of DNA from mature mRNA in a test tube (in vitro).

• ****Exons are the coding regions of DNA, while introns are non-coding regions.

• cDNA: Inserted into a bacterial plasmid.

• Transformation: Plasmid containing cDNA is introduced into a bacterial cell.

• Protein Expression: Bacteria express the cDNA, producing the encoded protein.

• Therapeutic Proteins: This method is used to produce insulin, growth hormone, cytokines, and other therapeutic proteins.

Regulatory Gene in recombinant DNA

• Regulatory Genes: Included in plasmids to control the expression of the inserted target gene.

• Inducers: Molecules (e.g., sugars like lactose or arabinose, or metal ions like iron, copper, or zinc) that trigger the regulatory gene.

• Mechanism: The inducer binds to the regulatory protein, causing a conformational change that allows the regulatory gene to be transcribed and translated. This, in turn, allows the target gene to be transcribed and translated, leading to protein production.

• Importance:

  • Protein Production: Essential for controlling when and how much of the target protein is produced. This is particularly important in industrial settings where large-scale protein production is required.

  • Gene Expression Studies: Crucial for studying gene expression in plant and animal models. Researchers can use inducers to turn genes on and off at specific times to understand their function and regulation.

Transforming Bacterial Cell

• Transformation: The process by which a cell incorporates foreign DNA (e.g., a plasmid) into its genome. Cells that have taken up foreign DNA are said to be "transformed."

• Transformed Cells: These cells acquire new characteristics because they can now express the genes carried by the foreign DNA. For example, a bacterium that takes up a plasmid containing an antibiotic resistance gene becomes resistant to that antibiotic.

• Genetic Transformation: Another term for the process of introducing foreign DNA into cells.

• Natural vs. Artificial Transformation: Transformation can occur naturally (some bacteria can naturally take up DNA from their environment) or artificially (scientists use various techniques to force cells to take up DNA). In the context of recombinant DNA technology, we are generally talking about artificial transformation.

Natural Transformation of Bacterial cell

• Natural Genetic Exchange: Bacteria have several natural mechanisms for exchanging DNA. This is important because it allows for genetic diversity even though they reproduce asexually (e.g., by binary fission).

• Evolution and Antibiotic Resistance: Natural genetic exchange is a major driver of bacterial evolution, particularly in the spread of antibiotic resistance. Bacteria can acquire genes conferring resistance from other bacteria.

• Bacterial Competence: This refers to a bacterium's ability to take up DNA from its environment or other bacterial cells. This is a key factor in the spread of antibiotic resistance genes.

• Antibiotic Resistance Research: Because antibiotic resistance is a serious and growing threat to public health, bacterial competence is a significant area of ongoing research. Understanding how bacteria become competent and exchange resistance genes is crucial for developing strategies to combat antibiotic resistance.

Artifical Transformation of Bacterial cell

Two common methods of artificial bacterial transformation:

• Heat Shock:

  1. Bacteria and plasmids (both recombinant and non-recombinant) are placed in a cold calcium ion solution.

  2. The temperature is rapidly increased (heat shock).

  3. This disrupts the bacterial cell membrane, allowing plasmids to enter.

• Electroporation:

  1. Bacteria and plasmids are subjected to an electrical current.

  2. The current temporarily alters the cell membrane, allowing plasmids to enter.

• Transformation Efficiency: Both methods have low efficiency. Few bacteria are successfully transformed. Some may take up non-recombinant plasmids, and many will not be transformed at all or will die during the process.

• Sufficient Transformation: Even though the efficiency is low, a small number of successfully transformed bacteria is enough to grow a large culture. This is because bacteria reproduce rapidly.

Selection and Screening of transformed bacteria

• Plasmid Vector Characteristics: Essential for identifying transformed bacterial cells with recombinant plasmids containing target DNA.

• Antibiotic Resistance Gene: Plasmid vectors often contain a gene for antibiotic resistance (e.g., ampicillin resistance) to select for transformed cells.

• Reporter Gene: Plasmid vectors may include a reporter gene that produces a detectable phenotype, such as a colored product, to confirm successful transformation.

• Selection and Identification: Antibiotic resistance allows for the selection of transformed cells, while the reporter gene aids in visual identification of successful recombinants.

Selection of transformed bacteria

• Transformation Identification: To determine which bacterial cells have been transformed with the antibiotic resistance gene, bacteria are grown on nutrient agar plates containing the antibiotic (e.g., ampicillin).

• Optimum Conditions: Plates are incubated at 37°C, the optimal temperature for bacterial growth and colony formation.

• Survival of Transformed Cells: Only bacteria that have taken up the plasmid (either recombinant or non-recombinant) will survive, as they contain the ampicillin resistance gene (ampR).

• Elimination of Non-Transformed Cells: Bacteria without the plasmid (and thus lacking the ampR gene) will be killed by the antibiotic.

• Outcome: Surviving colonies indicate successful transformation with the plasmid containing the antibiotic resistance gene.

Screening for bacteria transformed with Recombinant Plasmids

• lacZ Gene Function: The plasmid carries the lacZ gene, which codes for an enzyme that breaks down X-gal, producing a blue product.

• Non-Recombinant Plasmid: Bacteria with a non-recombinant plasmid (intact lacZ gene) produce blue colonies on agar plates.

• Recombinant Plasmid: If target DNA is inserted into the lacZ gene, its expression is disrupted, and the enzyme is not produced. Bacteria with recombinant plasmids form white colonies.

• Visual Identification: Blue colonies indicate non-recombinant plasmids, while white colonies indicate successful insertion of target DNA (recombinant plasmids).

• Culturing Recombinant Bacteria: Bacteria with recombinant plasmids are cultured with nutrients to replicate and produce the protein (e.g., insulin) encoded by the target DNA.

• Reference: Figure 4.2.13 illustrates the differentiation between blue and white colonies.

Protein products of Recombinant DNA

• Recombinant Protein Production: Recombinant DNA is introduced into bacteria or eukaryotic cells to synthesize proteins.d

• Types of Proteins Produced:

  • Hormones

  • Cytokines

  • Enzymes

  • Vaccines

• Therapeutic Applications:

  • Epidermal Growth Factor: Used in burn treatment to improve skin graft survival.

  • Interleukin-2: Used in cancer treatment.

  • Antibodies: Employed in immunotherapy.

  • Vaccines: Developed against various viruses.

  • Insulin: Produced for diabetes treatment, safer and more effective than animal-derived insulin (e.g., from pigs).

• Advantages Over Traditional Methods: Safer and more effective than using proteins purified from other organisms (e.g., growth hormone from human pituitary glands).

• Industrial Applications:

  • Enzymes like amylase, lipase, protease, and cellulase are used in:

    • Food processing

    • Textile industry

    • Detergent additives.

Genetically modified and Transgenic organisms

• Humans have used selective breeding for tens of thousands of years to produce animals and plants with more useful or attractive characteristics.

• Organisms displaying desired traits were selectively bred to pass those traits to offspring.

• Historically, selective breeding was limited to traits already present in a species' gene pool.

• Modern advancements allow DNA manipulation techniques to directly alter an organism's genetic material.

• Genetic manipulation offers potential benefits but raises questions about its biological, social, and ethical implications.

Gene Editing with CRISPR-Cas9

• Gene editing allows organisms to have their genome modified through directed mutation (mutagenesis) or newer technologies like CRISPR-Cas9.

• CRISPR-Cas9 is a gene editing technology that can cut DNA at specific locations.

• CRISPR stands for "clustered regularly interspaced short palindromic repeats."

• CRISPR refers to segments of DNA containing short, repetitive sequences interspersed with unique DNA sequences.

• This technology enables precise modifications to an organism's genetic material.

Function of CRISPR-Cas9 in Bacteria

• CRISPR arrays in bacteria contain fragments of viral DNA captured from previous viral invasions.

• When the same virus attacks again, the bacterium uses the CRISPR arrays to transcribe RNA sequences complementary to the virus's DNA.

• These RNA sequences guide the system to the virus's DNA.

• The viral DNA is cut and disabled using endonuclease enzymes.

• The specific enzyme responsible for cutting the viral DNA is called Cas9, a CRISPR-associated protein.

• This mechanism acts as an adaptive immune system in bacteria, protecting them from viral infections.

Application of CRISPR-Cas9 in editing Genomes

• The CRISPR-Cas9 system can edit eukaryotic genes by combining the Cas9 enzyme with guide RNA (gRNA).

• Scientists artificially create gRNA, which contains a short sequence complementary to the target DNA.

• The gRNA directs the Cas9 enzyme to the specific target site in the DNA.

• Cas9 cuts the DNA at the target location.

• Researchers utilize the cell's natural DNA repair mechanisms to modify the DNA.

• DNA can be altered by:

  • Repairing base pair deletions or insertions associated with known mutations.

  • Inserting new DNA sequences.

• This process allows precise genetic modifications in eukaryotic cells.

• Figure 4.3.2 illustrates the CRISPR-Cas9 gene editing mechanism.

• Research with CRISPR-Cas9 in humans has begun, focusing on:

  • Editing cancer genes.

  • Modifying genes in embryos.

• Limitations of the Cas9-gRNA system:

  • The success rate for editing genes or cells is not well understood.

  • Recent findings show that not every position in the gRNA needs to match the target DNA, leading to off-target (unintended) edits.

  • Research on CRISPR-Cas9 efficiency is inconclusive, with success rates varying widely:

    • For three different genes: 13% to 43%.

    • For producing transgenic mice embryos: 2% to 88%.

• Bioethical concerns:

  • Editing germline cells (eggs or sperm) raises ethical issues, as changes will affect all cells in the resulting embryo and be passed to future generations.

  • Editing embryos may result in not all cells carrying the edited gene, but germline edits affect the entire organism.

  • The use of reproductive cells for CRISPR-Cas9 studies is currently illegal in most countries, but patent rights to the technology are being contested by companies.

• Technical challenges:

  • Most tested gRNA sequences are only around 20 nucleotides long, increasing the likelihood of unintended edits elsewhere in the genome.

  • This raises the risk of crucial genes being unintentionally modified.

• Future outlook:

  • Due to the risks and limitations, it will likely be many years before CRISPR-Cas9 is routinely used in humans.

Genetically Modified and Transgenic Organisims in Agriculture

• Over the last few decades, techniques have been developed to alter an organism's genome and transfer genes from one organism to another.

• Organisms with altered genetic material are called genetically modified organisms (GMOs).

• The universality of the DNA code allows genes transferred from one organism to another to express the same protein as in the original organism.

• This enables the transfer of desirable characteristics from one organism to another that lacks those traits.

• Transgenic organisms are GMOs that have had a gene from another species (called a transgene) inserted into their genome

• Applications of transgenic organisms:

  • Used in agriculture to increase crop productivity.

  • Provide resistance to diseases in plants and animals.

Genetically Modified Animals

• Transgenic cows and sheep are used in agriculture for:

  • Improved fertility.

  • Enhanced meat production.

  • Better milk quality and yield.

  • Improved wool quality and yield.

• The use of genetically modified (GM) farm animals has not expanded as much as GM plants, possibly due to detrimental effects of some modifications, such as:

  • Genes promoting growth may cause altered skeletal growth, arthritis, and heart or kidney problems.

• Regulation of GM animals:

  • In Australia, GM animals are not approved for human consumption.

  • In 2015, the United States approved genetically modified Atlantic salmon for human consumption.

• Details of GM Atlantic salmon:

  • Contains a gene from another salmon species and a promoter sequence from a pout fish.

  • This modification allows the salmon to eat year-round, not just in warm water, leading to faster growth rates and earlier harvest.

  • 99% of GM salmon are sterile, reducing the risk of interbreeding with wild salmon if they escape.

  • This is the first GM animal approved for human consumption in the USA.

Genetically Modified Plants

• GM crops are used in agriculture to:

  • Increase crop productivity.

  • Provide resistance to insect predation.

  • Prevent diseases.

• Examples of GM crops in Australia:

  • Insect-resistant GM cotton has been grown since 1996.

  • Herbicide-tolerant GM canola was approved for commercial production in Victoria in 2008.

• Regulation of GM organisms in Australia:

  • The Office of the Gene Technology Regulator (OGTR) assesses all GM animals or plants before they are used for research, agricultural, or commercial purposes.

Techniques for producing transgenic plants

• Challenges in gene transfer to plants:

  • The plant cell wall can limit the introduction of foreign genes.

• Common method for gene transfer:

  • Uses Agrobacterium tumefaciens, a soil bacterium that naturally transfers a plasmid into plant cells.

• Role of Agrobacterium tumefaciens:

  • Normally causes crown gall disease by transferring a plasmid with tumor-inducing genes.

  • Scientists use a recombinant plasmid (vector) that carries a desired gene but lacks tumor-inducing genes.

• Process of gene transfer:

  1. The recombinant plasmid is introduced into Agrobacterium cells.

  2. Transformed Agrobacterium is cultured with plant cells, transferring the recombinant plasmid into the plant cells.

  3. Transformed plant cells are grown in tissue culture to develop new plants.

  4. These plants are transplanted into the field as transgenic crops.

• Figures:

  • Figure 4.3.6 illustrates the natural plasmid transfer by Agrobacterium.

  • Figure 4.3.7 shows the process of growing transgenic crops from transformed plant cells.

Increased Crop Productivity-(salt tolerant wheat)

• Soil salinity is a major issue in Australian agriculture, causing:

  • Osmotic water loss from roots and tissues due to high sodium salt levels.

  • Cell stress from altered sodium and potassium ion ratios.

• Natural salt tolerance in plants:

  • Salt-tolerant plants protect themselves by:

    • Preventing sodium entry into cells.

    • Storing salt in vacuoles.

    • Pumping sodium out of cells.

  • Molecular biologists have identified the genes controlling these traits.

• Genetic modification for salt tolerance:

  • Scientists from the University of Adelaide introduced a salt-tolerant gene from an Australian native plant into wheat.

  • This gene codes for a protein that removes sodium from leaves, allowing normal water movement from roots to leaves.

  • Results:

    • Improved grain yield in salty soils.

    • No negative impact on grain yield in normal soil.

• Benefits of salt-tolerant wheat:

  • Expands the geographical range for wheat production in Australia and other salinity-affected regions.

  • Addresses the growing need for food production as the global population increases.

• Figure 4.3.8 illustrates the improved grain yield of salt-tolerant wheat.

Disease Resistance

• Irish Potato Famine (1840s):

  • Caused by the plant pathogen Phytophthora infestans, which leads to late blight in potato and tomato crops.

  • Resulted in 1 million deaths and 1.5 million people emigrating from Ireland.

• Ongoing impact of Phytophthora infestans:

  • Remains a major problem for potato and tomato crops, nearly 180 years after its first documentation.

  • Controlled with fungicides, but still causes significant financial losses.

• Genetic solution to late blight:

  • Researchers in the United Kingdom identified a resistance gene in American black nightshade, a wild relative of the potato.

  • The gene was successfully inserted into potatoes, creating genetically modified (GM) potato crops resistant to late blight.

  • This eliminates the need for fungicides to control the disease.

• Significance of the breakthrough:

  • Provides a sustainable solution to a long-standing agricultural problem.

  • Reduces reliance on chemical treatments and minimizes crop losses.