Genetic Technology & Genetic Therapy PDF

1/31/2025 What is a Clone? Week 11 – Part A Clone Genetic Technology & An identical copy of a DNA segment, whole cell or...

1/31/2025 What is a Clone? Week 11 – Part A Clone Genetic Technology & An identical copy of a DNA segment, whole cell or complete organism, derived from a single ancestor Genetic Therapy Cloning The process of producing identical copies of molecules, cells, or organisms based on eBook Chapter 11 How do we create a Clone? Carrot cloning Plants can be cloned from single cells 1950’s individual carrot cells were grown in the lab – Charles Steward Cells grew into a ball of undifferentiated cells (callus) In different media calluses grew into normal carrots 1  1/31/2025 Plant cloning Animal cloning Not as easy as for plants! Plants can be cloned from a variety of tissues Traditional cloning involved mating two organisms with Leaves, roots, stem desirable traits to get offspring All plant cloning involves de-differentiation (forgetting The offspring with the best combination of traits were what type of cell they are) then used as parents Then re-differentiation (specialising) using different Happening in agriculture for centuries! media Artificial Selection Examples of historical animal cloning Newer Cloning Methods Embryo Splitting Variation on the natural occurrence of twins First done in 1902 – Hans Spemann Artificial “twinning” process More recently - collect egg from female and fertilise in vitro Allow embryo to develop into 4-8 cells Separate all cells  4-8 embryos Implant into surrogates All offspring are clones 2  1/31/2025 Embryo Splitting Newer Cloning Methods Nuclear Transfer Sperm injected into More technically difficult egg cell for fertilisation Larger number of cloned offspring Egg cell Nucleus from egg cell removed Nucleus from another cell inserted First accomplished in 1952 by Robert Briggs and Thomas King on the Northern Leopard Frog Early Nuclear Transfer Newer Cloning Methods Nuclear Transfer 1958 – John Gurdon used intestinal cells to create a clone Early embryo Mammalian eggs smaller and more difficult to work Embryo cell – Embryo nucleus with nucleus removed implanted into donor egg 1975 – J. Derek Bromhall first mammalian embryo created by nuclear transfer Normal growth and development Donor egg – nucleus removed 3  1/31/2025 Nuclear Transfer – Dolly the Sheep Nuclear Transfer – Dolly the Sheep 1997 - First nuclear transfer using adult DNA (Somatic Cell Nuclear Transfer) Nuclear fusion rather than injection Low success rate – 277 fusions 29 embryos 13 surrogates 1 full term pregnancy What happened to Dolly? Nuclear Transfer these days Lived a pampered life Egg cell taken from donor, nucleus removed Had lambs of her own Adult cell taken from animal to be cloned Euthanased at 6½ years of age Nucleus from adult cell injected into empty egg Arthritis, lung tumour, early ageing Egg ”reprograms” the nuclear material Telomere shortening or cell differentiation Encouraged to divide by electric shock Implanted into surrogate Much higher success rate than nuclear fusion Routinely used in agriculture – sheep, cows, goats, pigs Dolly and her lamb Bonnie Dolly in the Edinburgh Museum 4  1/31/2025 Nuclear Transfer these days Cloning genes Does not involve cloning whole organisms Recombinant DNA Technology Produces clones of DNA molecules Transfer of genes between species Used to find genes and map them Identifies carriers of genetic disorders May be used in gene or genetic therapy DNA cloning Restriction Enzyme DNA cutting Requires three things: Bacterial natural defense enzymes 1. A way to cut the DNA consistently Hunt out specific sequences in DNA and cut both 2. A carrier molecule to transfer the DNA into your strands cell of choice Usually 4-8bp recognition sequences 3. A host cell into which to transfer the DNA Some enzymes make a blunt cut Some enzymes create a sticky end Prevents infection of foreign DNA Hundreds of Restriction Enzymes (REs) Each has it’s own specific recognition sequence 5  1/31/2025 Restriction Enzyme cutting patterns Sticky ends are used in cloning Recognition and Recognition sequence is identical on both strands (5’3’) cleavage Enzyme sequence Cleavage pattern Source organism Sticky ends can bind in a complementary fashion Ligases can stick overlapping DNA together EcoRI Escherichia coli Haemophilus Sticky ends HindIII influenzae Bacillus BamHI amyloliquefaciens Staphylococcus Sau3A aureus Blunt ends Haemophilus HaeIII aegypticus Combining DNA Vectors carry DNA to the host cell Genetically engineered plasmids Plasmids are found naturally in bacteria Small, circular DNA in the cytoplasm Get copied when bacteria divide Bacteria can make use of the genes carried on the plasmid 6  1/31/2025 Creating a vector Ligation is not always successful Plasmid and DNA cut with same RE Ligation reaction Correct DNA self- vector ligates Plasmid reforms, or formed joins other plasmids Host cells replicate the DNA Selectable Markers Once a vector is produced it is inserted into a host cell Something which helps us tell the difference between for replication transformed and non-transformed bacteria 6 The result? Usually antibiotic resistance Recombinant plasmids enzyme 5 The that carry foreign DNA. Only those cells that have been transformed will grow in DNA These plasmids are fragments introduced into host the presence of the antibiotic and the cut cells, which divide to 1 A restriction enzyme plasmid form clones. cuts a specific base 3 The DNA are mixed. sequence everywhere fragments have The sticky it occurs in DNA. sticky ends. ends of different fragments that base- pair are 4 The plasmid bonded by 2 The same enzyme DNA ligase. DNA also has cuts the same sticky ends. sequence in plasmid DNA. 7  1/31/2025 Do we have to clone all the DNA? Polymerase Chain Reaction (PCR) Nope Better way to copy DNA sequences for study Many techniques are available to allow you to target Technique of DNA replication targeting only the DNA specific genes for cloning you want to study Less clones need to be produced to find your gene of Requires several key components: interest DNA – something to copy Saving time and money and lab space Buffers – to provide the best conditions dNTPs – A’s, C’s, G’s and T’s to make a new DNA strand with Primers – starting point for duplication Taq polymerase – enzyme for copying Polymerase Chain Reaction Polymerase Chain Reaction Primers Primers Short DNA fragments (~20bp) Target DNA Complementary binding to DNA flanking your target sequence 3’ 5’ Provide a free 3’ end to which Taq polymerase can bind and begin copying 5’ 3’ One primer per DNA strand 3’ ends of primers must point towards one another Target DNA 3’ 5’ 5’ 3’ 8  1/31/2025 Polymerase Chain Reaction Polymerase Chain Reaction Taq Polymerase Taq Polymerase Can withstand high temperatures Similar to DNA Polymerase III in our cells Target DNA Reads DNA underneath and pulls dNTPs out of solution to create a new complementary DNA strand 5’ Works in a 5’ to 3’ direction 5’ A C G T Polymerase Chain Reaction Polymerase Chain Reaction Temperature cycling is required in PCR Taq Polymerase Denaturation Target DNA Annealing Extension Multiple rounds of cycling gives exponential increase in ATG 5’ your target DNA Quickly builds up lots of copies for further research 5’ ACA A C G T 9  1/31/2025 PCR cycling PCR cycling Denaturation – 95oC Annealing – 50-65oC Heating the dsDNA (double-stranded), breaks hydrogen Primers bind in a complementary fashion to ssDNA bonds  two strands of ssDNA (single-stranded) Denaturation (95oC) Denaturation ssDNA Annealing (50-65oC) PCR cycling PCR cycling Extension – 72oC At the end of the first cycle we now have 2 double- Taq polymerase binds to the 3’ end of the primers, reads stranded pieces of DNA the DNA base on the single strand and pulls a Denaturation (95oC) complementary nucleotide out of the solution Reforms the double stranded DNA Annealing (50-65oC) Extension (72oC) 50-65oC 10  1/31/2025 PCR cycling PCR cycling Cycle 2 Cycle 3 Denaturation now creates 4 templates  4 dsDNA strands Denaturation creates 8 templates  8 dsDNA strands PCR is exponential PCR also increases non-target DNA Target DNA is between the primers Taq polymerase doesn’t know where to stop More and more target DNA over time 11  1/31/2025 PCR overview What can we use cloned sequences for? Generate animal models for disease Genetically engineer animals with disease causing mutations See what disease processes are occurring Transfer ideas and and findings to human situation https://www.youtube.com/watch?v=iQsu3Kz9NYo What can we use cloned sequences for? What can we use cloned sequences for? Generate animal models Create stem cells for medical treatment Animals that do not need to be dissected to see the Stem cells have the ability to turn into any cell type or organs and blood systems at work tissue Used to repair damaged or diseased tissues Take someone’s own cells to develop treatment 12  1/31/2025 What can we use cloned sequences for? What can we use cloned sequences for? Bring back extinct species Cloning livestock Requires DNA from the extinct animal To get better meat, milk, wool production A closely related species to be an egg donor and Pretty common undertaking surrogate mother All attempts so far have been unsuccessful Useful for preserving endangered species What can we use cloned sequences for? What can we use cloned sequences for? Plants to detect environmental changes Producing drugs When environment changes, the appearance of the Generate bacteria that will produce human products plant changes Plants can detect landmines 13  1/31/2025 What can we use cloned sequences for? Edible Vaccines Producing drugs Potato Banana Advantages: Advantages: Edible vaccines Easily transformed Don’t need cooking Easily propagated Inexpensive Stores for long periods without Grown widely in developing refrigeration countries Disadvantages: Disadvantages: Needs cooking which destroys Trees take 2-3 years to mature antigen Fruit spoils rapidly after ripening Tomato Rice Advantages: Advantages: Grow quickly Commonly used in Cultivate broadly Baby food High content of Vit A may boost High expression of antigen immune response Disadvantages: Disadvantages: Grows slowly Fruit spoils rapidly Requires glasshouse conditions What can we use cloned sequences for? What can we use cloned sequences for? Cloning pets Cloning pets With enough money and will this is possible Ethical issues – pet farms Though, they don’t always look the same Egg donors, surrogates, unwanted pups/kittens Rainbow Her clone CC (Carbon Copy) 14  1/31/2025 What can we use cloned sequences for? What can we use cloned sequences for? Cloning pets Cloning humans Generating species with novel appeal Ethical, legal and social concerns Therapeutic cloning Time to Kahoot! Genetic Therapy Can we use this technology to correct malfunctioning Go to: www.kahoot.it genes? Removing exons with disease-causing mutations, just Type in the access code that will appear on the like the natural process of alternative splicing screen Give yourself a nickname – nothing rude or you’ll be banned! 15  1/31/2025 Gene Expression Review What is Exon Skipping? A technique that can be used to either: o Remove disease-causing exons from the mRNA o Skip over additional exons so that the mRNA Pre-mRNA sequence makes sense Exon skipping Masks the exons from the splicing machinery Maintaining Reading Frame Maintaining Reading Frame mRNA codes for amino acids in 3bp codons Larger deletions can have similar effects A single deletion or insertion moves the codon down Codons can extend across exons one position in the sequence Deletion of part of a codon by deleting the exon also This can completely change the resulting protein causes reading frame changes produced Amino acid sequence 123 123 123 123 123 12 3 1231 23123 Met – Pro – Gly – Tyr --- Met – Pro – Val – Met --- 123 123123 123 123 12 Exon skipped 3123 1 Met – Pro – Leu – Stop 16  1/31/2025 Maintaining Reading Frame Normal Dystrophin Gene Expression Think of exons as fitting together like a puzzle Dystrophin gene is on chromosome X Some exons can be skipped, but it still fits together Contains 79 exons, ~3,600 amino acids Removing the wrong piece means the protein no longer Provides a structural link to maintain muscle integrity works Mutations in DMD produce Duchene and Becker Muscular Dystrophies Muscular Dystrophy Mutations Muscular Dystrophy Mutations Often a result of skipping or deletion of exons In Duchenne MD, the reading frame is changed In Becker MD, the reading frame remains intact though No functional protein produced  more severe the protein may not function as well as it should 17  1/31/2025 Exon skipping Exon Skipping Artificially skip exons with disease-causing mutations Using anti-sense oligonucleotides (AON) to mask exons Skip additional exons to return sequence to the correct from the splicing machinery reading frame 20-30bp, complementary binding Sense and Anti-sense Anti-sense oligonucleotides Two strands of DNA – Sense (coding) and Anti-sense Anti-sense oligos are identical to the anti-sense (template) (template) strand DNA Sense strand Anti-sense strand Pre-mRNA Pre-mRNA Anti-sense oligo 18  1/31/2025 Anti-sense oligonucleotides Exon Skipping Cover the splice recognition site, or Bind to exon recognition sites in the exons https://www.youtube.com/watch?v=nvHi2yo0oD8 Exon Skipping DMD therapy progress A number of exons can be targeted in DMD A number of exon skipping AONs have been produced Some must not be skipped though Show some promising results in studies AONs have been mostly well-tolerated Does not replace lost muscle, but slows progression May be suitable for 83% of DMD sufferers 19  1/31/2025 Week 11 – Part B Unit Review and Exam information UTEI evaluations of units and teaching are open! Check your student email for survey links Help us maintain good learning experiences for students Final Assessment Preparation Final Assessment Preparation Study Guide is on Canvas (Assignments) What to bring with you to the exam Final exam will contain two parts: Student ID card Part 1 – 50 Multiple Choice Questions Pens, pencils, eraser, ruler, sharpener Part 2 – 5 Long Answer Questions A clear water bottle You need to do BOTH parts You need to hand in your exam paper, MCQ sheet What NOT to bring to the exam and answer booklet at the end of the exam A watch You will have 2 hours Calculator Exam is worth 40% of your final mark Food 20  1/31/2025 On the Day of the Exam Final Exam – Part 1 Arrive at least 15-30 minutes early, especially if Covers all lecture content for the entire semester your exam is in a large venue 50 MCQs, worth 40 marks Turn off your mobile phone and place it, and your, Weekly quizzes are available on Canvas valuables in your bag Answers need to be filled in on a separate Multiple- Enter the venue in a single line and sit where told Choice Answer Sheet Fill in your exam information when asked to do so Make sure you know how to do this before the exam Leave your ID in the top right corner of the desk If you need anything, raise your hand Canvas > Assignments > Assessment Information > You will not be admitted after 1 hour How to fill in a Multiple-Choice Answer Sheet You cannot leave in the last 10 minutes Instruction Panel ECU’s Use pencil Multiple- Erase your answers fully if you change your mind Choice Answer Sheet Completely fill in the circles 21  1/31/2025 Student Information Panel Surname and Initials Write in CAPITAL letters, Has space for: one letter per box Surname Initials Follow vertically down Student Number and fill in the circle for Test Date that letter One letter per column Student Number and Date Unit Code Panel Write the numbers in, one May or may not be present number per box Use CAPITAL letters and numbers and fill out as Follow vertically down previously and fill in the circle for that number One letter or number per column One number per column Ignore the “Special Code” box if present 22  1/31/2025 Answer Section Final Exam – Part 2 Fill in the circle Five (5) long answer questions (not essays) corresponding to the YOU ONLY NEED TO DO TWO (2) answer you have chosen Covers content from across the entire semester in the question paper Eight (8) study areas have been provided for you in the Study Guide, the questions will be taken from Erase any mistakes and fill these areas in the correct answer Worth 40 marks, 20 marks each question You can use bullet points and diagrams Q1-40 on the front, Q41- Note – in the deferred exam there is only 2 questions, no choice 50 on the back Study Area 1 Study Area 2 Week 1 Lecture – Cells & Cell Division Week 3 Lecture – Cytogenetics: Karyotypes and Chapter 2 of your eBook Chromosomes Aberrations Chapter 5 of your eBook Topics to focus on: Topics to focus on: The Interphase stage of the cell cycle and what Karyotypes – what they are, how they are done, occurs in each step what information they can tell you and examples of The process of Mitosis and what occurs in each step when and how they are used The process of Meiosis and what occurs in each Polyploidy – details about what it is, how it occurs step Aneuploidy – details about what it is, how it occurs The differences between Mitosis and Meiosis and examples of common sex chromosome Aneuploidies 23  1/31/2025 Study Area 2 continued Study Area 3 Week 3 Lecture – Cytogenetics: Karyotypes and Week 5 Lecture – Development and Sex Determination Chromosomes Aberrations Chapter 6 of your eBook Chapter 5 of your eBook Topics to focus on: Topics to focus on: The male and female reproductive systems, key Explain the process of non-disjunction and outline structures of each the various outcomes depending on when it occurs Spermatogenesis – detail what happens at each Describe the various structural changes within chromosomes, including production of unbalanced step, how the sperm mature and move through the gametes reproductive system Oogenesis – detail what happens at each step and where these things occur Study Area 3 continued Study Area 4 Week 5 Lecture – Development and Sex Determination Week 6 Lecture – DNA Structure and Chromosomal Chapter 6 of your eBook Organisation Chapter 7 of your eBook Topics to focus on: The differences between spermatogenesis and Topics to focus on: oogenesis The experiments that led to discovery that DNA X-chromosome inactivation – details about why it is carries the genetic information necessary, how it happens and the effect it has on The contributions made by various researchers that the cells in a female allowed Watson and Crick to come up with their model of the structure of DNA 24  1/31/2025 Study Area 4 continued Study Area 5 Week 6 Lecture – DNA Structure and Chromosomal Week 7 Lecture – Gene Expression: From Genes to Organisation Polypeptides Chapter 7 of your eBook Chapter 8 of your eBook Topics to focus on: Topics to focus on: Details about Watson and Crick’s model of DNA Transcription, including the enzymes involved, the structure steps in the process and post-transcriptional DNA Replication – the enzymes involved and what modifications that turn pre-mRNA into mRNA each one does, including Okazaki fragments Translation, including the roles for the various types The key structures in the chromosomes and the of RNA and the steps in the process role for each Study Area 5 continued Study Area 6 Week 7 Lecture – Gene Expression: From Genes to Week 9 Lecture – Cancer Genetics Polypeptides Chapter 10 of your eBook Chapter 8 of your eBook Topics to focus on: Topics to focus on: Differences between familial and sporadic cancers Role of cell cycle checkpoints and what happens at Basic information about the various levels of each one protein structure Normal role of Tumour-Suppressor genes, what Details about the effect a mutation in the DNA changes lead to cancer, give an example sequence can have on the resulting protein, Normal role of Proto-oncogenes, what changes lead including the different types of mutations that can to cancer, give an example occur 25  1/31/2025 Study Area 6 continued Study Area 7 Week 10 Lecture – Cancer Genetics Week 10 Lecture – Genetic Technology and Genetic Chapter 10 of your eBook Therapy Chapter 12 of your eBook Topics to focus on: Normal role of DNA repair genes, what changes Topics to focus on: lead to cancer, give an example of one gene and its Details about animal cloning techniques function in DNA repair Details about restriction enzymes - what they are Discuss how cancer can be caused by chromosomal and the various ways they cut translocation and give an example Details about vectors – what they are, how we create them and possible ligation outcomes Polymerase Chain Reaction, the key components, their roles in the reaction, the temperature steps, what happens at each step Study Area 7 Study Area 8 Week 10 Lecture – Genetic Technology and Genetic Based on the laboratory activities that you have Therapy undertaken throughout the semester, which are Chapter 12 of your eBook covered in your SCH1133 Human Genetics Laboratory Workbook Topics to focus on: Be able to explain why PCR results in an exponential Topics to focus on: increase in your target DNA, but also a small Details about the process of karyotyping, including increase in non-target DNA what reagents are used and what each one of them Details about the technique of Exon skipping – what does. Details about the specific volumes of each it is, what it achieves, how it works and how it can reagent used is NOT necessary, just the steps taken be used in Duchenne Muscular Dystrophy and what role each of the reagents plays in the process 26  1/31/2025 Study Area 8 continued Study Area 8 continued Topics to focus on: Topics to focus on: Details about the genetic transformation of Details about the Polymerase Chain Reaction (PCR), bacteria, including the method and the advantages including information about the key ingredients in and disadvantages of this technique. Details about the reaction, what each one does, the specific volumes of reagents or incubation times temperatures steps taken when doing a PCR and are NOT required, merely the steps that we took what happens at each step. and what happens in each step. Explain electrophoresis and how we can use it to Explaining selectable markers and how we used separate DNA on the basis of size them in your transformation experiment 27