BIOL305 Fall 2024 Lecture 35: Studying Development and Disease PDF

Lecture 35: Studying development and disease Dr. Nayantara Govindrajan, PhD (she/her) [email protected] BIOL305 Fall 2024 Wednesday December 4th , 2024 1 Lecture Outcomes ▪ Learn ho...

Lecture 35: Studying development and disease Dr. Nayantara Govindrajan, PhD (she/her) [email protected] BIOL305 Fall 2024 Wednesday December 4th , 2024 1 Lecture Outcomes ▪ Learn how recombinant DNA technology is used to study development and disease ▪ Gain an understanding of DNA sequencing and gene cloning ▪ Familiarize yourself with the process of DNA amplification by PCR ▪ Learn how genetic engineering is used to create transgenic animals and for gene therapy 2 © 2017 Pearson Education, Inc. Biotechnology The technical application of biological knowledge and engineering for human purposes Commercial Industrial Health Includes: Recombinant DNA technology Genetic engineering Gene therapy Ibrahim Khairov, Wikimedia Commons, CC 4.0 3 Recombinant DNA technology Recombinant DNA: DNA combined from 2 or more sources DNA sequencing allows us to determine the precise sequence of bases in DNA Uses: Primers: initiate DNA synthesis Fluorescently labeled nucleotides: added one at a time in sequencing Complementarity Each time a modified nucleotide is added→ synthesis stops Enzyme: facilitates addition of nucleotides to growing DNA strand DNA polymerase: replicates DNA Gel electrophoresis: separates DNA fragments 4 © 2017 Pearson Education, Inc. DNA sequencing Step 1: Extracted DNA fragment is amplified Complementary by DNA polymerase. Millions of C TA base sequences A* copies of the fragment are mixed Primer G G C*A G G*CT*C with primers and labeled T A GC T A* nucleotides Sequence to T*A be analyzed G*T A Primers bind to a region in the DNA and + Enzyme act as a starting point for synthesis of a C*G T A new DNA strand C*C G T A Based on complementarity A*C C G T A In the first step, the sequence to be analyzed is mixed with primer, polymerase, and Now, fluorescent nucleotides are labeled nucleotides, producing short complementary DNA sequences, each ending with a labeled nucleotide. added one by one to the growing strand-DNA synthesis Based on complementarity Facilitated by the enzyme Final result → mixture of new DNA strands of varying lengths, each ending with a single, Synthesis can be performed with a modified, fluorescently labeled nucleotide normal or modified nucleotide. Each time a modified nucleotide is added, the synthesis stops. 5 © 2017 Pearson Education, Inc. DNA sequencing Fluorescent intensity A T G C C A* Step 2: A T G C C* A T G C* DNA strands are separated based A T G* on size by gel electrophoresis A T* A* DNA migrates through the pores present in a slab of gel, under the influence of an A photo of a DNA sequencing gel A T G C C A electric field In the second step, the short DNA The result is a printout of the DNA is negatively charged → moves electrophoresis sequences produced by sequence of the complementary towards positively charged electrode the first step are separated by gel and strand of DNA. then Smaller bands move faster in the gel analyzed by a laser. Final result is a gel with differently colored DNA strands at different locations in the gel Step 3: Laser scans the gel and reads the Complementary to the locations of the nucleotides original strand→ original strand sequence can be found Result: graphic display of nucleotides in the new strands, arranged by sizes of the strands 6 © 2017 Pearson Education, Inc. The Human Genome Project From 1990-2003, mapped the human DNA sequence for the first time ~90% Considered the greatest biological feat of the 20th century Used and optimized DNA sequencing technology Publicly available sequences of humans and other model organisms Themes and variations. Nat Rev Genet 2, 158–159 (2001). 7 https://doi.org/10.1038/35056081  Recombinant DNA technology: Gene cloning Palindromic Restriction sequence Recombinant DNA enzyme cut Cutting, splicing, and copying DNA Tools for manipulating DNA Restriction enzymes: cut DNA Restriction “cut” at specific sites, often enzyme cut palindromes Leaves single-stranded fragments that are complementary to each other “paste” DNA ligases: join fragments of DNA Plasmids: small circular pieces of DNA to which desired genes can be added and inserted into bacteria for amplification 8 © 2017 Pearson Education, Inc.  E. coli Human cell Example: EcoRI Plasmid DNA is isolated from enzyme recognizes DNA bacterial and human cells. Gene cloning GAATTC Bacterial DNA Both DNAs are cut with the Palindromic same restriction enzyme. sequences Human DNA containing gene Why the same restriction “cut” of interest enzyme? DNAs are mixed. Human fragments line up with DNA fragments plasmid by base pairing It will leave single-stranded of exposed single-strand regions. fragments that will be complementary to each other DNA ligase is added to “paste” connect human and Now we have a circular bacterial plasmid DNA together. plasmid containing human DNA Recombinant DNA Plasmids are absorbed Bacteria containing plasmid of by bacteria. interest will be chosen and cloned Bacteria containing the recombinant plasmids of interest are selected and cloned. Bacterial clones carrying copies of the human genes 9 © 2017 Pearson Education, Inc. Cloning DNA fragments: PCR Polymerase Chain Reaction Can be used to rapidly amplify DNA sequences to obtain millions of copies Reaction requires: DNA to be amplified (template) Primers: bind to DNA template and act as a starting point for synthesis Heat-stable DNA polymerase: synthesizes new complementary DNA strands Repeated heating and cooling cycles allow for rapid amplification of a sequence of DNA defined by the primers 10 © 2017 Pearson Education, Inc.  Sample of double-stranded DNA PCR DNA is unwound by gentle heating; the single strands will serve as templates for new strands. The single strands of DNA are mixed with primers able to bind to one end. Nucleotides and DNA polymerase are added. As the mixture cools, the primers pair with the ends of the template strands. Nucleotides are attached in sequence, and the DNA strands replicate. The number of double- stranded DNA molecules doubles. 11 © 2017 Pearson Education, Inc. PCR The mixture is reheated, unwinding the double-stranded DNA molecules again. The mixture is cooled again. Primers again pair with the ends of each strand, and nucleotides again are attached in sequence. Replication again doubles the number of double-stranded DNA molecules. 12 © 2017 Pearson Education, Inc. DNA Fingerprinting Identify the source of a DNA fragment Used in forensics, paternity testing, genealogy, evolutionary relationships (fossils) Jamie Luther, Flickr, CC 4.0 Based on the principle that between genes in DNA, there are long repeating sequences of base pairs called short tandem repeats or STRs 5-25 bps Number of times an STR repeats is highly variable between individuals Leon Brocard, Flickr, CC 4.0 Gene 1 Gene 2 13 DNA fingerprinting DNA is collected and then amplified using PCR DNA is cut with restriction enzymes Different individuals will have different lengths of STRs between the restriction cut sites Fragments formed after cutting by restriction enzymes will vary in size DNA is separated according to size by gel electrophoresis and fragment pattern is compared Produces a pattern of separation of fragments: DNA fingerprint 14 © 2017 Pearson Education, Inc.  The Human Organism Question break Code: 531703 Blood Blood There has been a break in at a Stain jewelry store. The investigative team collected some blood samples from broken glass and are matching it to 4 different Suspect 1 suspects. Based on the DNA fingerprint generated by STR testing, who do you think is the Suspect 2 culprit? A. Suspect 1 B. Suspect 2 Suspect 3 C. Suspect 3 D. Suspect 4 Suspect 4 15 © 2017 Pearson Education, Inc. Question break Blood Blood There has been a break in at a Stain jewelry store. The investigative team collected some blood samples from broken glass and are matching it to 4 different Suspect 1 suspects. Based on the DNA fingerprint generated by STR testing, who do you think is the Suspect 2 culprit? A. Suspect 1 B. Suspect 2 Suspect 3 C. Suspect 3 D. Suspect 4 Suspect 4 16 © 2017 Pearson Education, Inc. Genetic engineering to create transgenic organisms Transgenic organism: any organism that has been genetically modified to contain recombinant DNA Transgenic bacteria: Manufacture human proteins like insulin and growth hormone Transgenic plants: Increased resistance to freezing (tomatoes) Increased resistance to pests Golden rice: higher beta carotene Converted to vitamin A in the body, which can enrich diets Longer shelf life Edible vaccines Hepatitis B vaccine in potatoes, being researched in banana (does not have to be cooked) 17 © 2017 Pearson Education, Inc. Genetic engineering to create transgenic organisms Transgenic animals: trickier to generate: Animal cells don’t take up plasmids as easily Cloning animals from single cells is much harder How they are generated: Foreign DNA can be microinjected into fertilized eggs Agitate fertilized eggs in the presence of foreign DNA and tiny silicon needles Microinjection, CC 3.0, Wikimedia commons Successes: Bovine growth hormone (bGH): used to promote faster animal growth Study human diseases using mouse models- transgenic mice that overexpress proteins associated with Alzheimer’s disease 18 © 2017 Pearson Education, Inc.  DNA of Genetic engineering to create interest Plasmid transgenic organisms Goat ovum Inject plasmids containing human DNA into ovum nucleus. “Gene pharming”: introducing Implant fertilized ovum into a female for development. human genes into dairy animals in such a way that the human protein is Analyze the offspring’s DNA to produced in the dairy animal’s milk determine if it has incorporated the human DNA. Used to create anti-thrombin III in The DNA of interest is expressed and the goats, which can prevent blood clots protein is secreted into the milk. during surgery Collect milk. Milk containing the protein Separate milk proteins. Isolate the useful human protein. 19 © 2017 Pearson Education, Inc. Gene therapy Inserting human genes into human cells to correct or treat disease We know the gene mutations for diseases like cystic fibrosis and hemophilia → try to fix these using gene therapy Obstacles: Difficult to introduce genes into the appropriate target cells where the genes would normally be expressed Need effective means of delivering genes Genes may not be passed to offspring Corrective genes introduced into reproductive cells to stop the passing of defective genes to offspring 20 Gene therapy Normal gene Delivery of the desired gene to the patient cell is done by 1 vectors Retrovirus Incorporate normal gene into retroviral vector Transporters Include: ❑Nonviral: Liposomes: plasmids containing target gene are inserted into small fatty spheres Electroporation: electric field temporarily causes pores in the Cell taken 2 cell membrane → foreign DNA can enter from patient Infect patient’s cells with retrovirus vector Pros: no risk of infection, reduced risk of immune response Cons: less efficient , and can carry less DNA ❑Viral: Retroviruses, adenoviruses Human genes can be packaged in retroviruses, which can introduce genes into human cells Pros: efficient, can carry more DNA 3 Cons : may cause infections and trigger an immune response Inject retrovirus-infected Viruses are usually modified to remove the genes/proteins that cause cells back into patient immune response prior to injecting them in the body FDA approved the first cell-based gene therapy for sickle-cell anemia in 2023. Patient blood stem cells were modified to contain a gene (delivered by lentivirus) that produced a protein that did not form the sickle shape and carried oxygen 21 © 2017 Pearson Education, Inc. Biorender Review Recombinant DNA technology allows us to modify DNA DNA sequencing is the process which identifies the order of nucleotide bases in a gene, DNA fragment or entire organism DNA fragments can be amplified by PCR Genes can be cloned using restriction enzymes, and transferred to different organisms STR testing uses restriction enzyme digestion and PCR to identify unique patterns of STRs in individuals Genetic engineering creates transgenic organisms Gene therapy can be used to treat human disease 22 Practice Questions 1. You are performing DNA sequencing but mistakenly used an expired enzyme that no longer works. Which of the following will be true: A. Newly synthesized, fluorescently labeled DNA fragments will not be able to migrate through the gel B. Synthesis cannot begin due to the lack of primer binding C. Fluorescently labeled nucleotides cannot be added after primer binding 23 Practice Questions 2. Which of the following is the most critical factor to ensure successful amplification by PCR? A) The DNA template should be in large quantities to ensure effective amplification. B) The primers should be specific to the target region of the DNA to prevent non-specific amplification. C) The reaction temperature should be kept constant throughout the PCR cycles to ensure efficiency. 24 Practice Questions 3. Spider silk is used to make certain wound dressings and bandages. A scientist is trying to produce spider silk in goat milk by gene pharming. In 2-3 lines, explain how she would achieve this? 25 Practice Questions 1. You are performing DNA sequencing but mistakenly used an expired enzyme that no longer works. Which of the following will be true: A. Newly synthesized, fluorescently labeled DNA fragments will not be able to migrate through the gel B. Synthesis cannot begin due to the lack of primer binding C. Fluorescently labeled nucleotides cannot be added after primer binding 26 Practice Questions 2. Which of the following is the most critical factor to ensure successful amplification by PCR? A) The DNA template should be in large quantities to ensure effective amplification. B) The primers should be specific to the target region of the DNA to prevent non-specific amplification. C) The reaction temperature should be kept constant throughout the PCR cycles to ensure efficiency. 27 Practice Questions 3. Spider silk is used to make certain wound dressings and bandages. A scientist is trying to produce spider silk in goat milk by gene pharming. In 3-4 sentences, explain how she would achieve this? Isolate the silk gene from spiders, and insert it into a bacterial plasmid Inject the plasmid into a fertilized goat egg and analyse the resulting offspring to see if it expresses spider silk gene Collect the milk and isolate the spider silk protein from the offspring expressing the recombinant gene