Chapter 12: Biotechnology & Bioengineering - Lecture Notes PDF

12.1 Biotechnology & Bioengineering Bioengineering Biotechnology Combining math, Physically, chemically, or computers, engineering, molecularly altering an biology, chemistry and organism physics Designing new tissues, Cloning, transgenic medical...

12.1 Biotechnology & Bioengineering Bioengineering Biotechnology Combining math, Physically, chemically, or computers, engineering, molecularly altering an biology, chemistry and organism physics Designing new tissues, Cloning, transgenic medical devices, organisms prosthetics, artificial hearts, etc.  Genetic Engineering Genes are isolated, modified, and inserted into an organism – Same or different organism Made possible by recombinant DNA technology – Cut up DNA and recombine pieces – Restriction enzymes & vectors 12.2 Forensic Biotechnology Genetic Profiling (DNA Fingerprinting) Comparison of DNA between individuals for identification Most are rape cases (>2 out of 3) Looking for match between evidence and suspect How Genetic Profiling is Used Forensic cases -- matching suspect with evidence Paternity testing -- identifying father Historical investigations Missing persons investigations Mass disasters -- putting pieces back together Military DNA “dog tag” Convicted felon DNA databases Brief History of Forensic DNA Typing 1980 - Ray White describes first polymorphic RFLP marker 1985 - Alec Jeffreys discovers multilocus VNTR probes 1985 - first paper on PCR 1988 - FBI starts DNA casework 1991 - first STR paper 1995 - FSS starts UK DNA database 1998 - FBI launches CODIS database 2017 - FBI adds 7 loci to testing http://www.cstl.nist.gov/biotech/strbase/intro.htm Challenges for Genetic Profiling Incomplete or partial DNA Profiles due to  DNA is often degraded  Limited quantities  Inhibitors (contaminants) to PCR are often present  Evidentiary samples are often mixtures. http://www.councilforresponsiblegenetics.org/GeneWatch/GeneWatchPage.aspx?pageId=57  Forensic Biotechnology Focus on short tandem repeats (STR): shorter repeating nucleotides (3-7 bases long) Form from unequal crossing over during meiosis Compare sequences known to be variable between individuals (figure 12.1) FBI’s CODIS - Combined DNA Index System National DNA Index System (NDIS) Used for linking serial crimes and unsolved cases with repeat offenders launched October 1998 & Links all 50 states DNA profile in database consists of one or two alleles at the 13 CODIS Core Loci. The National DNA Index (NDIS) contains over 17 million offender profiles over 5.4 arrestee profiles and 1.3 million forensic profiles as of February 2024 FBI’s CODIS - Combined DNA Index System National DNA Index System (NDIS) Sometimes only partial profiles obtained:  For forensic DNA profiles, all 13 CODIS Core Loci must be attempted but at least 8 CODIS Core Loci combined with a match rarity of at least one in ten million are required for submission to and searching at NDIS.  For Missing Person and Unidentified Human Remains, all 13 CODIS Core Loci must be attempted.  Partial matches may cause familial DNA testing 13 CODIS Core STR Loci with Chromosomal Positions TPOX D3S1358 TH01 D8S1179 D5S818 VWA FGA D7S820 CSF1PO AMEL D13S317 D16S539 D18S51 D21S11 AMEL Sources of Biological Evidence Blood Semen Saliva Urine Hair Teeth Bone Tissue Polymerase Chain Reaction - PCR chromosome cell nucleus Double stranded DNA molecule Target Region for PCR Individual nucleotides  PCR - DNA Amplification 5’ 3’ 5’ 3’ Starting DNA Template 3’ 5’ 3’ 5’ Separate strands (denature) Add primers Forward primer (anneal) 5’ 3’ 5’ 3’ Make copies (extend primers) 5’ 3’ 5’ 3’ Reverse primer Learn about PCR https://www.youtube.com/watch?v=c07_5BfI DTw https://learn.genetics.utah.edu/content/labs/p cr/ Gel Electrophoresis Tool to separate STR fragments based on size. Samples are loaded into a gel and a current applied, DNA fragments move with current toward positive end of gel Smaller molecules move further than larger ones and gives a pattern  Gel Electrophoresis The pattern of DNA bands is compared between each sample loaded on the gel Running a gel Short Tandem Repeats (STRs)  Learn more about gel electrophoresis https://www.youtube.com/watch?v=GUXKQB knYQo https://www.youtube.com/watch?v=vq759wK CCUQ https://learn.genetics.utah.edu/content/labs/g el At bottom of page click on Can DNA determine a verdict?  12.3 Genetic Engineering – Transgenic Biotechnology Gene Splicing: Splicing genes from one organism into another to add new genes or alter genes Results in new form of DNA: Recombinant DNA Genetically modified organism (GMO) or transgenic organism Vectors A DNA molecule that replicates on its own in a host cell and can be used as a vehicle in the laboratory for replicating your DNA (or gene) of choice. – Plasmids: Circular DNA molecules often found in bacteria that are separate from chromosomal DNA and can self-replicate. – Viruses: Viruses contain genetic material but are not living. Host cells are required for their reproduction. The host cell uses the genetic instructions in the virus to make more viruses.  12.3 Genetic Engineering Basic Steps to Create a GMO 1.) Find & isolate gene of interest 2.) Decide on vector (plasmid or virus) 3.) Cut gene of interest and insert into vector. 4.) Introduce vector to new organism Human Insulin is made using this process One cell with the recombinant plasmid Then the single cell with many A fermentor used to grow This is the stepplasmids recombinant cloning takes place. recombinant bacteria when geneproduces The single trillions recombinant of like plasmid replicates within a cell. cells with recombinant plasmid – and the human insulin gene.  Transgenic Plants Resistant to herbicides Insect resistance  Golden Rice or Frankenfood? Scientists transferred daffodil genes into rice Rice with beta-carotene may help prevent vitamin A deficiencies Opponents fear unforeseen consequences of creating genetically modified organisms overview  GMO Benefits Useful products: 1.) production of medically useful proteins 2.) research into gene regulation and control 3.) production of more nutritious, insect resistant crops with higher yields less herbicide and insecticide used 4.) development of new species of garden plants  GMO Concerns Possible safety and disease issues with eating the foods Only been used for aprx. 20 years – no independent research in U.S. Must be “generally recognized as safe”; no differences in nutritional content No labelling required in U.S. Banned in Europe and Australia Current GMO Crops Current GMO Crops Genetically engineered animals Transgenic Animals  Transgenic Animals Agriculture – Breeding and Quality – cows with more milk, sheep with more wool, pig/cattle with more meat Industry – Goats that make spider silk Medicine – Goats – antithrombin – Rabbits – IL-2 for immune system – Cattle – collagen for skin grafts Franken-fish Cloning: There are three different types of artificial cloning: Gene cloning, reproductive cloning and therapeutic cloning. 1. Gene cloning produces copies of genes or segments of DNA. 2. Reproductive cloning produces copies of whole animals. 3. Therapeutic cloning produces embryonic stem cells for experiments aimed at creating tissues to replace injured or diseased tissues. 12.4 Cloning: Reproductive Cloning Reproductive cloning uses a donor egg but replaces its nucleus with a somatic cell nucleus. Egg contains all the organelles, proteins, enzymes etc. but the DNA of the offspring will be a clone of the somatic cell donor Also known as Somatic Cell Nuclear Transfer (SCNT) Cloning 101 12.4 Reproductive Cloning: SCNT  Why clone animals? To answer questions of basic biology For pharmaceutical production To satisfy our desires (e.g. pet cloning) For herd improvement  What about cloning humans? Currently the American Medical Association is against the development of human clones, however, some advocacy groups believe it could be useful in certain circumstances (i.e., infertility). SCNT & Therapeutic Cloning Patients somatic cells are harvested and used to create stem cells that are a genetic match Used to create stem cells to grow replacement tissues or even inject into patient to treat the disease  12.4 Reprogramming Cells – Regenerative Medicine Embryonic stem cells (ESCs) Adult (somatic) stem cells Induced pluripotent stem cells (iPS cells)  Reprogramming Cells – Regenerative Medicine Excellent Website with Stem Cell Information 12.5 Human Gene Therapy Genetic diseases are not contagious Gene therapy is the correction of defective genes for disease – Addition of a functional gene copy – Replacement of defective gene – Repair of defective gene – Shut off defective gene Need to know what the problem is: is the gene a mutant gene? Is it under expressed? Is too much protein being made? Gene Therapy Intro Gene Therapy & Cell Therapy Patient’s cells removed and treated with a viral vector containing the normal gene, cells multiply and are returned to the patient. Ex vivo In vivo  Human Gene Therapy Even with the human genome fully sequenced, it is still not easy to manipulate Viruses used to insert genes into cultured human cells Very difficult to get modified genes to work where and how they should To stop a mutant gene from working it must be changed – usually inserting a mutation into the promoter will turn it off – some scientists use transposons or viruses  Viruses are the vectors of choice for animal cells. They can accept larger amounts of DNA than plasmids. When the virus reproduces within the animal cell, it also reproduces the foreign gene that it carries. Gene therapy using viral vectors  Human Gene Therapy The challenge: 1.) Scientists must find a way of taking the DNA with the corrected gene and putting it into the cell 2.) The corrected DNA must be made a part of the cell’s DNA so that a.) it is passed on with each cell division b.) it doesn’t interfere with other genes c.) the corrected DNA can be transcribed the cell as needed 3.) Cells containing the corrected DNA must be reintroduced to the patient Gene Therapy Gene Therapy for Diabetes Mellitus Diabetes mellitus: insert the insulin gene into other body cells such as liver or stomach Convert non-hormone producing cells of pancreas into beta cells (cells that make insulin) iPS cells investigated to be reprogrammed into beta cells Gene therapy that code for immune suppressive proteins introduced into the pancreas to stop the immune system from destroying beta cells diabetes Problems with Gene Therapy 1.) understand the mechanics and exact nature of the problem 2.) understand the mechanisms and find out how the problem can be resolved 3.) identify possible side effects from treatment 4.) work through the ethical and moral issues that must be decided upon by professionals and clients 5.) work out financial considerations 6.) deal with hurdles: governmental approval of new medical procedures Gene Editing: CRISPR Genome Editing with CRISPR Pharmacogenomics Genetically based medication Dosage based on patient’s genetics  Pharmacogenomics Enzymes genetically encoded and determine how quickly drugs are metabolized. Pharmacogenomics Simulated Activity