ANSC20010 Genetics and Biotechnology Section 6 Past Slides PDF

ANSC20010 Genetics and Biotechnology: Section 6 Spring Trimester, 2023-24 ANSC20010 Section 6: DNA Biotechnology Campbell 11th Edition chapter 20 Transgenic tobacco plant expressing the luciferase gene from a firefly 1 Assessment and mark-to-grade conversion scales for ANSC20010 (Genetics and Biotec...

ANSC20010 Genetics and Biotechnology: Section 6 Spring Trimester, 2023-24 ANSC20010 Section 6: DNA Biotechnology Campbell 11th Edition chapter 20 Transgenic tobacco plant expressing the luciferase gene from a firefly 1 Assessment and mark-to-grade conversion scales for ANSC20010 (Genetics and Biotechnology) Spring Trimester 2023-24 Continual assessment MCQ1 and 2: 25 questions - no negative marking, pass mark 50%. 5% mark/grade weighting each. End-of-Trimester MCQ: 50 questions - no negative marking, pass mark 50%. 90% mark/grade weighting each. 2 Learning Objectives for Section 6 Understand how DNA (and proteins) are analysed in the laboratory:  DNA extraction.  An overview of DNA cloning (molecular cloning/gene cloning).  The polymerase chain reaction (PCR) and PCR applications.  DNA sequencing technologies and applications. How DNA can be manipulated in the laboratory to perform practical tasks or create useful products (DNA-based biotechnology): Transgenic animals, Cloning animals.  Transgenic plants. Genome editing. 3 1 ANSC20010 Genetics and Biotechnology: Section 6 Spring Trimester, 2023-24 What is Biotechnology? UN Convention on Biological Diversity (1992): “Any technological application that uses biological systems, living organisms, or derivatives thereof, to make or modify products or processes for specific use.” Biotechnology (DNA technology) – “the manipulation of organisms or their biological components (DNA or proteins) to perform practical tasks or to provide biomedical products” The application of DNA biotechnology to specific biological, medical or agricultural studies is often called genetic engineering. 4 A timeline of human-directed plant biotechnology Francis D., et al. (2017) Challenges and opportunities for improving food quality and nutrition through plant biotechnology. Curr. Opin. Biotechnol. 44, 124-9. 5 Biotechnology: some recent major milestones 1953 Discovery of 3D structure of DNA 1966-1968 Genetic code is deciphered 1973 1st recombinant bacterium constructed 1986 PCR developed 1977 DNA sequencing 1996 1st cloned mammal 1990 Human Genome Project (HGP) initiated 2007 - onwards High-throughput genome sequencing 2001 1st draft of HGP 2012 CRISPR-Cas9 Genome editing 2003 HGP completed 6 2 ANSC20010 Genetics and Biotechnology: Section 6 Spring Trimester, 2023-24 1970s: the dawn of DNA manipulation technologies Paul Berg – Nobel prize winner 1980 “We developed a general way to join two DNAs together in vitro; in this case, a set of three genes responsible for metabolizing galactose in the bacterium E. coli was inserted into the SV40 DNA genome. That work led to the emergence of the recombinant DNA technology ” 1970s – DNA can be manipulated in vitro to enable researchers to modify specific genes and transfer them between different organisms (genetic engineering). Recombinant DNA – any DNA molecule that has been generated in vitro and contains DNA sequences from two or more distinct DNA molecules, often from different species. 7 DNA extraction and purification Sample collection Cell pellet Hypersaline Lysis of cells Solution Treatment with organic solvents Add aqueous layer to ethanol Precipitate DNA and dissolve in buffer solution 8 DNA cloning generates large amounts of pure DNA from specific segments of a genome How can geneticists examine single genes from a genome of interest? Difficulty in examining single genes:  Naturally-occurring DNA is very long (many kilobases) and can include many discrete genes (genomic DNA).  Eukaryotic genes occupy only a small portion of the genome. To work directly with specific genes, scientists need to prepare multiple, identical copies of a gene: can be achieved through DNA/gene cloning. 9 3 ANSC20010 Genetics and Biotechnology: Section 6 Spring Trimester, 2023-24 DNA cloning often involves the use of bacteria and their plasmids Plasmid: small DNA molecule replicating independently of the bacterial chromosome (i.e., the bacterial genome). Plasmid sizes can range between 1 kb–200 kb and often carry genes for antibiotic resistance. Plasmids are maintained at stable number from cell to daughter cell. Used as cloning vectors into which foreign genes are inserted for subsequent cloning or expression in bacterial cells. Bacterium Bacterial chromosome Plasmid 10 An overview of how bacterial plasmids (cloning vectors) are used to clone (i.e. make many copies) genes Cell containing gene of interest Bacterium Bacterial chromosome Plasmid Gene of interest Chromosome Gene inserted in plasmid [recombinant DNA] Recombinant bacterium (only one recombinant molecule taken up by each cell) 11 Recombinant bacterium Produces a clone of identical cells, each containing the recombinant molecule. Clone with identified. Recombinant molecule is replicated along with the vector during bacterial cell division. gene-of-interest Copies of gene isolated and transferred to other organisms Gene for pest resistance inserted into a plant genome. Copies of protein product isolated Gene used to alter bacteria to clean up toxic waste. Protein used to dissolve blood clots. Human growth hormone treats stunted growth. 12 4 ANSC20010 Genetics and Biotechnology: Section 6 Spring Trimester, 2023-24 Applications of gene cloning: human health Using laboratory techniques to produce pharmaceutical products. Escherichia coli Recombinant human insulin 13 Applications of gene cloning: agriculture Manipulating the genomes of plants to produce crops to increase production. Example: Creating plants that carry a gene conferring resistance to insect pests. Bt (Bacillus thuringiensis) corn Carries a bacterial gene that encodes a toxin, which is harmful to specific insect pests. 14 Applications of gene cloning: industry Creating microorganisms in laboratories that can be used to clean contaminated environments. Example: recombinant bacteria used to clean toxic waste at sea. 15 5 ANSC20010 Genetics and Biotechnology: Section 6 Spring Trimester, 2023-24 Gene cloning was made possible by the discovery of restriction enzymes (restriction endonucleases) Restriction enzymes – enzymes that cut or ‘cleave’ genomic DNA at a limited number of specific sites. Originally identified by the capacity of certain bacterial strains to “restrict” the growth of bacteriophage viruses on petri dish culture. Cut at specific DNA sequences called restriction sites. ~400 different restriction enzymes available (many can be purchased from commercial companies). Naturally occurring in bacteria – protect bacteria from invading organisms by cutting up their DNA (e.g., bacteriophage viruses). Bacteria protects their own DNA from restriction by methylating adenine and cytosine residues (addition of -CH3 groups). 16 Naming of restriction endonucleases EcoRI restriction enzyme Bacteria: Escherichia coli Strain: RY13 First restriction enzyme recognised from this strain. to be 17 Restriction enzymes cleave duplex DNA at particular nucleotide sequences Most restriction sites are palindromic: the DNA sequence reads the same on the top or bottom DNA strand (in a 5'-to-3' direction). Palindrome examples in English: AND MADAM DNA or RACECAR. The recognition site for EcoRI (note: always cuts the same sequence): 5'G A A T T C 3' 3'C T T A A G 5' 5' G A A T T C 3' 3' C T T A A G 5' 18 6 ANSC20010 Genetics and Biotechnology: Section 6 Spring Trimester, 2023-24 Most restriction enzymes produce overhangs (‘sticky ends’) Staggered overhangs (aka ‘sticky ends’) generated: very useful for DNA cloning. 19 Some restriction enzymes produce ‘blunt ends’ Some endonucleases result in the formation of blunt ends (no overhang) e.g., SmaI restriction enzyme: 5'- CCCGGG – 3' 3'- GGGCCC – 5' SmaI 5'-CCC 3'-GGG GGG-3' CCC-5' 20 Recognition sequences and cleavage sites of representative restriction endonucleases Enzyme Source Recognition Sequence (5' - 3') GAATTC CTTAAG EcoRI Escherichia coli strain RY13 HincII Haemophilus influenzae strain Rc GTPyPuAC CAPuPyTG HindIII Haemophilus influenzae strain Rd AAGCTT TTCGAA HpaII Haemophilus parainfluenzae CCGG GGCC AluI Arthobacter luteus AGCT TCGA N.B. Bases surrounding cleavage sites are shown in blue Py = pyrimidine (cytosine/thymine); Pu = purine (adenine/guanine) 21 7 ANSC20010 Genetics and Biotechnology: Section 6 Spring Trimester, 2023-24 Restriction enzymes https://youtu.be/pDHCHa1C85Y 22 Example: cloning a bovine gene into a bacterial plasmid 1) Purify plasmid (cloning vector) DNA and bovine DNA lacZ (lactose breakdown) Bovine DNA containing gene of interest Restriction site ampR (ampicillin resistance) 2) Cut both DNA samples with the same restriction enzyme 23 Example: cloning a bovine gene into a bacterial plasmid Cloning vector: DNA molecule that can carry foreign DNA into a cell and replicate there. Plasmid is replicated as bacteria multiply. The resulting bacterial colony will contain multiple copies (clones) of the foreign DNA. Two important genes in the cloning vector: ampR confers host resistant to ampicillin (antibiotic). lacZ encodes -galactosidase, which hydrolyses (breaks down) lactose. Plasmid has single restriction site for the enzyme within the lacZ gene. 24 8 ANSC20010 Genetics and Biotechnology: Section 6 Spring Trimester, 2023-24 Example: cloning a bovine gene into a bacterial plasmid 5' X X X X X X X X G A A T T C X X X X X 3' 3' X X X X X X X X C T T A A G X X X X X 5' EcoRI restriction enzyme cuts plasmid DNA EcoR1 “sticky” ends + AAT T C ? ? ? ? ? ? ? ? ? ? G G ? ? ? ? ? ? ? ? ? ?C T TAA Bovine gene sequence of interest produced by cutting bovine genomic DNA with same enzyme (EcoR1) 25 Example: cloning a bovine gene into a bacterial plasmid 3) Mix the DNAs; they join by basepairing - some plasmids join with the fragment of interest. 4) Add DNA ligase (shown as ‘glue’) to bond DNA covalently. Non-functional lacZ gene (split in two) 5) Population of recombinant plasmids is the result. Bovine DNA fragment containing gene of interest 26 Example: cloning a bovine gene into a bacterial plasmid ligase ligase AAT T C ? ? ? ? ? ? ? ? ? ? G G ? ? ? ? ? ? ? ? ? ?C T TAA DNA molecules (plasmid DNA plus the bovine gene insert) stick together via base-pairing and DNA ligase joins the molecules covalently to seal the strands. X X X X X X X X GAAT T C ? ? ? ? ? ? ? ? ? ? GAAT T C X X X X X X X X X X X X X C T TAAG ? ? ? ? ? ? ? ? ? ? C T TAAG X X X X X Recombinant DNA plasmid. This can be inserted into a bacterial cell. The bacterial cell components transcribe and translate bovine DNA and produce a functional bovine protein that can be isolated and purified. 27 9 ANSC20010 Genetics and Biotechnology: Section 6 Spring Trimester, 2023-24 Creating recombinant DNA molecules is facilitated by DNA ligase DNA ligase – an enzyme that ligates (i.e., joins together) two separate DNA molecules. In cloning, DNA ligase is used to join ‘foreign’ DNA to plasmid DNA to create a recombinant plasmid. ‘Seals’ the sticky ends together by creating a continuous sugarphosphate backbone. Acts as a ‘molecular glue’ during DNA cloning. 28 Creating recombinant DNA molecules is facilitated by DNA ligase 29 Example: cloning a bovine gene into a bacterial plasmid 3) Mix the DNAs; they join by basepairing - some plasmids join with the fragment of interest. 4) Add DNA ligase (shown as ‘glue’) to bond DNA covalently. Non-functional lacZ gene (split in two) 5) Population of recombinant plasmids is the result. Bovine DNA fragment containing gene of interest 30 10 ANSC20010 Genetics and Biotechnology: Section 6 Spring Trimester, 2023-24 Cloning a gene with bacterial plasmid https://youtu.be/piGOuud3f40 31 Example: cloning a bovine gene into a bacterial plasmid 6) Put plasmids into lacZ- bacteria via transformation (uptake of DNA from culture medium). 7) Clone bacteria - plate cells onto medium with ampicillin and X-gal. white Identify cells containing recombinant plasmids by ability to grow in ampicillin and their white colour. Non-recombinant plasmid has a functional lacZ gene and metabolises Xgal giving a blue product. blue 32 Petri dish with blue and white E. coli colonies. Blue colony cells contain – galactosidase activity because they have plasmids without foreign DNA inserts. The white cells have plasmids with inserted DNA. 33 11 ANSC20010 Genetics and Biotechnology: Section 6 Spring Trimester, 2023-24 The components of an artificial plasmid vector used for DNA cloning https://youtu.be/KRpik9mNRm0 34 The polymerase chain reaction (PCR) A laboratory thermocycler used for performing PCR 35 PCR is a method to make many copies (i.e. amplify) of a specified DNA sequence from a genome of interest 36 12 ANSC20010 Genetics and Biotechnology: Section 6 Spring Trimester, 2023-24 What exactly is the polymerase chain reaction (PCR)? PCR is a method for making many copies of a specific segment of DNA (amplify a specific region of the genome). Very efficient and performed completely in vitro. PCR can be thought of as a ‘molecular photocopier’ for DNA. Target 37 What exactly is the polymerase chain reaction (PCR)? Six components are required for PCR: Component 1: heat-resistant DNA polymerase (Taq polymerase). Component 2: a supply of deoxynucleotides (dATP, dCTP, dGTP, dTTP). Component 3: two oligonucleotide primers (short-stretches of nucleotides ~20 in length) that bind specifically to the DNA sequences flanking the gene or the genomic region of interest. Component 4: a buffer solution containing magnesium ions (Mg++). Component 6: target DNA (usually genomic DNA) from the species of interest. 38 Components of the polymerase chain reaction (PCR) 1. Taq polymerase: heat-stable DNA polymerase enzyme isolated from thermophilic Thermus aquaticus, a bacterial species that lives at ~90°C in hot springs. Taq DNA polymerase enzyme is stable at 95°C for long periods (normal DNA polymerases are denatured at high temperatures and then have no activity). 2. Oligonucleotide primers: two primers are complementary to approximately 20 nucleotides of the DNA sequence that flanks the target region and bind specifically to each side of the target (remember DNA polymerases add nucleotides to pre-existing nucleotide chains, so primed DNA template is required for DNA synthesis). 39 13 ANSC20010 Genetics and Biotechnology: Section 6 Spring Trimester, 2023-24 Components of the polymerase chain reaction (PCR) 3. Free nucleotides (dNTPs): provide building blocks for newly synthesised strands (dATP, dCTP, dGTP and dTTP) 4. Target DNA (Genomic DNA) from the species/sample of interest. 5. PCR buffer solution: all PCR components are mixed together in the presence of a buffer (solution with a suitable pH and salt concentration) that contains magnesium chloride (MgCl2). 40 DNA polymerase: an enzyme that replicates DNA Synthesises new DNA from pre-existing DNA template. Always moves in a 5'-to-3' direction. Adds nucleotides to the 3' end of the newly synthesised DNA. Priming: DNA polymerases can only add nucleotides to a pre-existing double-stranded nucleotide chain that is ‘primed’ through complementary base-pairing with the DNA template. During replication, the single stranded DNA template must be primed for DNA synthesis. 41 DNA polymerase in vitro Nucleotides Primer 5’ 3’-OH G OH C OH T OH A OH DNA polymerase 3’ DNA polymerase 5’ Single-stranded DNA template 42 14 ANSC20010 Genetics and Biotechnology: Section 6 Spring Trimester, 2023-24 Taq DNA polymerase is a heat-stable DNA polymerase that is essential for PCR Old Faithful, Yellowstone Park, USA – source of thermophilic Thermus aquaticus 43 Components of the polymerase chain reaction (PCR) Free nucleotides Taq polymerase A T G C 5' 3' 3' 5' Single-stranded oligonucleotide primers (~ 20 nucleotides) Purified DNA containing target sequences (usually genomic DNA) Target sequence (amplicon) 44 Molecular basis of the PCR 5' 3' At 94°C DNA strands separate and become denatured (melted) 3' 5' 5' 3' At 55°C primers bind to target 3' 5' 45 15 ANSC20010 Genetics and Biotechnology: Section 6 Spring Trimester, 2023-24 Molecular basis of the PCR 3' 5' 5' 3' Taq polymerase At 72°C Taq polymerase extends newly synthesised complementary strands Taq polymerase 3' 5' 5' 3' Two daughter molecules 46 The PCR Many PCR cycles 47 The temperature stages in a PCR amplification 2-3 mins 94°C Melt 72°C Extension 55°C Anneal 20°C 30 s 30 s 30 s 30 s 30 s 30 s 30 s 30 s 30 s 30 s 30 s 30 s 30 s 30 s 30 s 30 s 30 s Start Cycle 0 N copies Cycle 1 2N copies Cycle 2 4N... Cycle 3 8N... Cycle 4 16N... Cycle 5 32N... Cycle 6 64N... 48 16 ANSC20010 Genetics and Biotechnology: Section 6 Spring Trimester, 2023-24 Theoretical yield of PCR amplicon molecules per cycle of PCR CYCLE MOLECULES 1 2 2 4 3 8 4 16 5 32 6 64 7 128 8 256 9 512 10 1,024 49 Theoretical yield of PCR amplicon molecules per cycle of PCR CYCLE MOLECULES 11 2,048 12 4,096 13 8,192 14 16,384 15 32,768 16 65,536 17 131,072 18 262,144 19 524,288 20 1,048,576 50 Theoretical yield of PCR amplicon molecules per cycle of PCR CYCLE MOLECULES 21 2,097,152 22 4,194,304 23 8,388,608 24 16,777,216 25 33,554,432 26 67,108,864 27 134,217,728 28 268,435,456 29 536,870,912 30 1,073,741,824 51 17 ANSC20010 Genetics and Biotechnology: Section 6 Spring Trimester, 2023-24 Theoretical yield of PCR amplicon molecules per cycle of PCR CYCLE MOLECULES 31 2,147,483,648 32 4,294,967,296 33 8,589,934,592 34 17,179,869,184 35 34,359,738,368 36 68,719,476,736 37 137,438,953,472 38 274,877,906,944 39 549,755,813,888 40 1,099,511,627,776 52 Theoretical yield for a PCR amplification reaction (assuming 100% amplification efficiency) 𝑋 𝑋 2 Where: Xn = number of PCR products after cycle n X0 = initial number of PCR target sequences 53 The polymerase chain reaction (PCR) https://youtu.be/2KoLnIwoZKU 54 18 ANSC20010 Genetics and Biotechnology: Section 6 Spring Trimester, 2023-24 Important features of the polymerase chain reaction (PCR) The amplified target region of the genome is termed the amplicon. PCR is very useful for rapidly generating large quantities of a specific sequence. It takes approximately 2–3 hours for 30–40 cycles of a typical PCR reaction. The amplified DNA can be purified and used in downstream applications (e.g., DNA sequencing). 55 Advantages of the polymerase chain reaction (PCR) PCR is very powerful: starting with a single target molecule, after 25 cycles the theoretical yield is 225 = 3.4 × 107 amplicons. Very small amounts of sample can be used, and target DNA does not have to be good quality (biological material from forensic samples, degraded material, ancient DNA etc.). Can design automated high-throughput systems and process thousands of samples per day. The cost per sample is low. 56 Visualising DNA using gel electrophoresis 57 19 ANSC20010 Genetics and Biotechnology: Section 6 Spring Trimester, 2023-24 Visualising DNA using gel electrophoresis How can we visualise DNA molecules in the laboratory? Use a technique termed gel electrophoresis. All macromolecules (DNA, proteins) have an electric charge and they will move in an electric field. DNA can be loaded on a gel matrix (usually agarose, a gelatinous polysaccharide purified from red algae). If an electric current is passed through the gel, the DNA migrates. The rate of migration is determined by:  the strength of the electric field.  the size of the DNA fragment/s in base pairs. 58 Electrophoresis: fractionation and analysis of biological macromolecules -ve -ve Largest +ve +ve Smallest 59 Making an agarose gel for electrophoresis Agarose gel matrix is generally used for larger molecules with big differences in size. Polyacrylamide gel matrix is generally used for smaller molecules with small differences in size. 60 20 ANSC20010 Genetics and Biotechnology: Section 6 Spring Trimester, 2023-24 Making an agarose gel for electrophoresis Dissolve agarose in electrophoresis buffer. Add ethidium bromide and pour into mould. Insert a plastic ‘comb’ to make wells for DNA samples. Leave the gel mould and gel mixture to solidify (approx. 1 hour). Agarose gel mould with plastic combs for DNA samples 61 DNA sample preparation for agarose gel electrophoresis The loading buffer contains sucrose or glycerol to increase density and causes sample to ‘drop’ into the well. A ‘tracking’ dye is used to track the progress of the DNA as it migrates across the gel. The dye molecules also migrate from the negative electrode (cathode) to the positive electrode (anode). A ‘DNA size ladder’ (contains DNA fragments of known size) is electrophoresed simultaneously for convenient sizing of sample DNA fragments. 62 Loading a gel for agarose gel electrophoresis Put the gel in a tank of electrophoresis buffer (contains a solution of conductive electrolytes that generate an electric field between the -ve and +ve electrodes. Load the samples into the wells and include size ladder in one lane. 63 21 ANSC20010 Genetics and Biotechnology: Section 6 Spring Trimester, 2023-24 Running a gel for agarose gel electrophoresis 64 Running a gel for agarose gel electrophoresis: shorter DNA molecules migrate more rapidly towards the anode Mixture of DNA molecules of different sizes Power source – Cathode Anode + Gel 1 Power source – + Longer molecules 2 Shorter molecules 65 Electrophoresis of DNA fragments (animation) -ve electrode 500 bp 300 bp 200 bp +ve electrode 66 22 ANSC20010 Genetics and Biotechnology: Section 6 Spring Trimester, 2023-24 67 Viewing the DNA bands in the gel Ethidium bromide intercalates between the strands of double-stranded DNA. Fluoresces orange when excited by ultraviolet (UV) light. 68 DNA bands on a gel visualised using ethidium bromide: ANSC30100 Applied Biotechnology 2014 practical results 1500 bp 1000 bp 750 bp 500 bp 250 bp 1500 bp 1000 bp 750 bp 500 bp 250 bp 69 23 ANSC20010 Genetics and Biotechnology: Section 6 Spring Trimester, 2023-24 Minisatellite tandem repeat DNA profiling for genetic identification: PCR amplification and gel electrophoresis Individual 1 (12/9) Ch1A DNA profiles generated from PCR-amplified fragments Ch1B 1 Ch1A 2 3 4 Individual 2 (9/9) Ch1B Ch1A Individual 3 (4/8) Ch1B Individual 4 (14/9) Ch1A homozygous Ch1B 70 Minisatellite tandem repeat DNA profiling for genetic identification: multiple minisatellite loci 71 The amelogenin gene (encodes tooth enamel) sex-test in cattle: PCR and gel electrophoresis For Class I: X chromosome 280 bp For Rev Class II: Y chromosome Deleted 217 bp Rev Females will have a 280 bp PCR product (amplicon). Males will have a 280 bp amplicon and a 217 bp amplicon. 72 24 ANSC20010 Genetics and Biotechnology: Section 6 Spring Trimester, 2023-24 An example of amelogenin gene sex-test results for beef samples 500 bp 400 bp 300 bp 200 bp 73 DNA sequencing 74 DNA sequencing: Fred Sanger – inventor of the Sanger dideoxy" chain-termination method in 1977 The first reliable method to sequence the nucleotides in a segment of DNA was developed by Fred Sanger (Nobel Prize 1980 and he also won an earlier Nobel Prize in 1958 for developing methods for determining protein structure – particularly insulin). 75 25 ANSC20010 Genetics and Biotechnology: Section 6 Spring Trimester, 2023-24 The Sanger dideoxy" chain-termination method for sequencing DNA molecules Involves synthesising in vitro DNA strands that are complementary to a template DNA strand. The in vitro sequencing reaction mix contains:  Template DNA (cloned or PCR amplified DNA).  Deoxyribonucleotide triphosphates [dNTPs]: dATP, dTTP, dCTP, dGTP (building blocks of DNA that have a free 3' OH group).  DNA polymerase enzyme. Dideoxyribonucleotides triphosphates [ddNTPs]: ddATP, ddTTP, ddCTP, ddGTP. 76 Structure of a nucleotide and dideoxynucleotide dNTPs: (dATP, dCTP, dGTP, dTTP) ddNTPs: (ddATP, ddCTP, ddGTP, ddTTP) 77 The Sanger dideoxy" chain-termination method for DNA sequencing using radioactively labelled primers Involves synthesising in vitro DNA strands that are complementary to a template DNA strand. Uses DNA polymerase to make many new DNA strands. But synthesis is interrupted at each base by incorporation of ddNTP to create strands that differ in length by one base. 78 26 ANSC20010 Genetics and Biotechnology: Section 6 Spring Trimester, 2023-24 Reading DNA sequence from an X-ray film autoradiogram: the film is fogged by the radiolabelled DNA bands on gel Read the sequence of the complementary strand to the template strand by reading upwards. Sequence read from the autoradiograph. 5’-TAT GGC GTG ACA CCT AAA-3’ The template strand. 3’-ATA CCG CAC TGT GGA TTT-5’ Template sequence is the reverse complement of this. 79 The Sanger dideoxy DNA sequencing method https://youtu.be/6ldtdWjDwes 80 Automated Sanger DNA sequencing technology 81 27 ANSC20010 Genetics and Biotechnology: Section 6 Spring Trimester, 2023-24 Automated Sanger DNA sequencing technology https://youtu.be/SRWvn1mUNMA 82 Automated Sanger DNA sequencing technology: DNA sequence chromatogram trace file 83 High-throughput sequencing (HTS) Illumina sequencing system (sequencing by synthesis) 18 terabases (Tb) per run (3 days) 2.2 petabases (Pb) per year Can sequence ~14,000 human genomes to 30× coverage per year This system can sequence complete human genome for ~$1,000 www.illumina.com/systems/hiseq-x-sequencing-system.ilmn 84 28 ANSC20010 Genetics and Biotechnology: Section 6 Spring Trimester, 2023-24 Applications of DNA sequencing: Genome sequencing of important agricultural species Rice (2002) Cow (2009) Pig (2012) 85 Applications of DNA sequencing: Genome sequencing of extinct species 86 Applications of DNA sequencing: Genome sequencing of extinct species Aurochs in Lascaux Cave Paintings (painted 17,000 BP) Park et al. (2015) Genome sequencing of the extinct Eurasian wild aurochs, Bos primigenius, illuminates the phylogeography and evolution of cattle. Genome Biol. 16, 234. 87 29 ANSC20010 Genetics and Biotechnology: Section 6 Spring Trimester, 2023-24 Ancient admixture/gene flow from British aurochs Kerry cattle Highland cattle 88 Transgenic animals and plants Herman – the first transgenic bull. He carried in his genome the human lactoferrin gene (i.e., a gene from another species artificially introduced into his genome). 89 What are transgenic organisms? Organisms with genomes that have been altered by the artificial introduction of a gene from another species. Transgenic animals are genetically modified organisms (GMOs). The genome of a genetically modified organism has been altered by the introduction of genetic material from another species. Transgene is the term used for the ‘foreign’ gene inserted into the host genome. Transgenesis/genetic engineering – the process of creating transgenic animals. 90 30 ANSC20010 Genetics and Biotechnology: Section 6 Spring Trimester, 2023-24 Examples of transgenic animals that have been produced 91 92 The genome of transgenic organisms are altered in a way that is heritable When creating transgenic animals/plants, the transgene can be transferred in the host genome by being: 1. Physically integrated into the genome – i.e. transgene is incorporated into a chromosome 2. Introduced as a persistent genetic entity: The use of artificial chromosomes. BACs (bacterial artificial chromosomes): a DNA construct consisting of DNA sequences from bacterial plasmids. When introduced into cells they behave like chromosomes. 93 31 ANSC20010 Genetics and Biotechnology: Section 6 Spring Trimester, 2023-24 Methods used to generate transgenic animals 1) Microinjection of foreign DNA into embryo pronuclei. A pronucleus refers to the nucleus of a sperm or ova during the process of fertilisation, after the sperm enters the ovum, but before they fuse. 2) Nuclear transfer: Replacement of oocyte nucleus with nucleus of transgenic diploid donor cell (very useful for livestock). 94 Creating transgenic animals: microinjection of DNA into the pronucleus Promoter e.g. from lactoglobulin gene for expression in mammary gland Gene of interest Microinjection of recombinant DNA into male pronucleus Ova Superovulating (multiple ovulations) egg donor In vitro fertilisation Implant into foster recipient Offspring (1-5% transgenic, 95-99% nontransgenic). Transgenic offspring identified with PCR. 95 Microinjection of a transgene into one of the pronuclei of a pig oocyte 96 32 ANSC20010 Genetics and Biotechnology: Section 6 Spring Trimester, 2023-24 Creating transgenic animals: microinjection of DNA into the pronucleus Transgenic offspring identified Gene of interest (GOI) expressed in mammary tissue – GOI protein is secreted in milk Can isolate large quantities of GOI protein from milk 97 Creating transgenic animals: nuclear transfer - replacement of oocyte nucleus with nucleus of transgenic donor cell Insert foreign DNA Cell donor Cell culture 100% transgenic Clonal selection Fusion by electricity Cell division Ova Enucleated ova Superovulating egg donor Pseudopregnant recipient Remove nucleus 98 Nuclear transfer versus pronuclear microinjection Nuclear transfer is the best current technology for creating transgenic animals. Donor cells are cultured and are transferred into enucleated oocytes. Resulting animals have the same nuclear genome as the donor cells plus the transgene. Can quickly generate many transgenic offspring from one valuable founder. Key advantage of nuclear transfer: use of cultured transgenic cells as nuclear donors - transgenic efficiencies of 100% (1-5% for pronuclear microinjection). 99 33 ANSC20010 Genetics and Biotechnology: Section 6 Spring Trimester, 2023-24 Useful biological products produced by transgenic animals Gene product (protein) Tissue plasminogen activator (TPA) Function Dissolves blood clots Prevents strokes Human Growth Factor Treat pituitary dwarfism Human blood clotting factor VIII Treat haemophilia Human insulin Treat diabetes Bovine growth hormone Improved cattle yields Platelet-derived growth factor Treatment of skin ulcers 100 Procedures that do not involve transgenesis Certain procedures are sometimes mistakenly thought to be genetic engineering. All of the examples below do not involve the introduction of DNA from a different species.  Surrogacy: female implanted with an unrelated embryo.  Artificial insemination: artificial placement of male gametes into female reproductive tract for fertilisation.  In vitro fertilisation: male and female gametes fused in vitro and zygote placed back into female reproductive tract for gestation.  Transfer of non-genetically modified somatic cell nuclei into enucleated oocytes (e.g., cloning of Dolly the sheep). 101 Dolly was not transgenic—she did not carry a transgene in her genome Dolly the sheep – the first cloned mammal. Dolly was cloned using nuclear transfer technology using a somatic cell from the mammary gland of a donor animal. As Dolly did not carry a gene from a different species in her genome she was not transgenic. 102 34 ANSC20010 Genetics and Biotechnology: Section 6 Spring Trimester, 2023-24 Cloning using undifferentiated embryo cells and nuclear transfer (NB. Dolly was cloned from an adult somatic cell) 103 Mammalian cloning – big business? 104 Genetic engineering of plants Traditionally, genetic improvement of plants involved selective breeding experiments. Selective breeding involves choosing plant with a desirable trait and allowing it to produce offspring (often involves self-fertilisation).  Labour-intensive.  Time consuming.  Reduction of genetic diversity.  Cross-breeding of plants restricted to closely-related species. Recombinant DNA technologies have allowed researchers to overcome many of these problems using transgenesis of agriculturally important plant species. 105 35 ANSC20010 Genetics and Biotechnology: Section 6 Spring Trimester, 2023-24 Plant genetic engineering using the Ti plasmid of Agrobacterium tumefaciens Agrobacterium tumefaciens (soil bacterium) infects plants causing crown galls Ti plasmid (Tumour-inducing) ~ 200 kb A small section of Ti plasmid (T DNA) integrates into plant genome. Recombinant plasmids can be engineered into plant cell genomes via A. tumefaciens Regenerate whole plants using cell culture of the transgenic plant cells. 106 Plant genetic engineering using the Ti plasmid of Agrobacterium tumefaciens https://youtu.be/L7qnY_GqytM 107 Plant genetic engineering using the Ti plasmid of Agrobacterium tumefaciens T DNA with restriction site Ti plasmid DNA containing gene of interest A. tumefaciens Restriction enzyme and DNA ligase Plant with new trait Chromosomal DNA Recombinant Ti plasmid Introduce into plant cells in culture Inserted T DNA carrying new gene Regeneration of plant 108 36 ANSC20010 Genetics and Biotechnology: Section 6 Spring Trimester, 2023-24 Examples of practical applications for transgenic plants 1) Agronomic traits Herbicide resistance (e.g., Glyphosate resistance - Roundup Ready biggest selling herbicide in the US). Insect-resistance (e.g., Bt insecticidal proteins - kill European corn borer). Pathogen-resistance: viral, bacterial, fungal etc. Abiotic stress: drought, cold, heat and salt tolerance. Yield traits (photosynthesis, nitrogen fixation…) Post-harvest qualities: food processing - gluten and dough quality, shelf life etc. 109 Examples of practical applications for transgenic plants 2) Nutraceuticals – food that has medicinal effect on human health. ‘Golden Rice’ – contains genes from daffodils and bacteria that biosynthesise beta-carotene in rice (helps vitamin A deficiency). Rice with additional iron – rice containing the soya bean ferritin gene – ferritin encodes a protein that facilitates iron storage. Wheat that biosynthesises extra folic acid (prevent spina bifida). 110 ‘Golden rice’ - engineered to produce beta-carotene, a precursor of Vitamin A 111 37 ANSC20010 Genetics and Biotechnology: Section 6 Spring Trimester, 2023-24 CRISPR-Cas9 gene editing: a word processor for genome engineering 112 CRISPR-Cas9 gene editing: a word processor for genome engineering https://youtu.be/ZImVkl8QTW8 113 CRISPR-Cas9 gene editing: a word processor for genome engineering 114 38 ANSC20010 Genetics and Biotechnology: Section 6 Spring Trimester, 2023-24 ANSC20010 End-of-semester MCQ examination Two-hour paper-based MCQ examination. 90% of the marks contributing to the overall grade for the ANSC20010 module (continual assessment online Blackboard MCQs contribute 10% of marks). 50 questions with five multiple choice answers for each question. Marking scheme:  Each correct answer carries 2.0 marks.  No negative marking is applied.  An unanswered question will not be penalised. 115 ANSC20010 End-of-semester MCQ examination Ensure you use a HB pencil and mark the required information and answers properly - use horizontal tick marks. Make sure you enter your student number as numbers and as horizontal tick marks (also enter your student number and seat number on the question booklet). Do not leave two or more answers ticked; you will not receive any marks for this question - use an eraser if required. MCQ question booklet has pages for rough work and must be handed back with the MCQ answer sheet. Calculators are permitted and you will need a scientific calculator for many of the questions. Further information: www.ucd.ie/students/assessment/mcq.html 116 Choice of pencil for filling out MCQ answer sheets: always use a HB pencil 2H: incorrect - too light HB: correct – right shade 117 39 ANSC20010 Genetics and Biotechnology: Section 6 Spring Trimester, 2023-24 2H: incorrect - too light 118 HB: correct – right shade 119 40