Designer Genes (C)KAREN LANCOUR 2019 Greg Palmer National Bio Rules Committee Chairman [email protected] er.net National Event Supervisor Event Rules 2019 DISCLAIMER This presentation was prepared using draft rules. There may be
some changes in the final copy of the rules. The rules which will be in your Coaches Manual and Student Manuals will be the official rules. Event Rules 2019 BE SURE TO CHECK THE 2019 EVENT RULES FOR EVENT PARAMETERS AND TOPICS FOR EACH COMPETITION LEVEL Designer Genes (C)
Content Molecular Genetics, Biotechnology plus General Principles DNA structure, function and replication Types of RNA, transcription and post-transcription modifications Translation and Universal Code Control of Gene Expression Organelle DNA - mitochondrial inheritance DNA technologies such as cloning, sequencing,
analysis, fingerprinting, and PCR, gene therapy Trinucleotide repeats and other disorders Gene Therapy, Bioethics, and Epigenetics Next Generation Sequencing Platform Process Skills - observations, inferences, predictions, data analysis, and calculations TRAINING MATERIALS
Training Power Point content overview 4 Training Handout overview, general principles, molecular genetics & biotechnology 2 Practice Activities molecular genetics & biotechnology sample problems & lab evaluations Sample Tournament sample problems with key Event Supervisor Guide prep tips, event needs, and scoring tips Internet Resource & Training CDs on the Science Olympiad website at www.soinc.org under Event Information
Biology-Earth Science CD, Genetics CD as well as the Division B and Division C Test Packets are available from SO store at www.soinc.org GENERAL PRINCIPLES OF GENETICS for Designer Genes Students need to review the general principles of genetics These areas are covered in the Heredity event in Division B so
these event materials are a good review Most event supervisors include some general principles and problems on their competitions Emphasis Scheme General Principles plus General Principles of Classical Genetics plus Regional and State Tournament Topics National Tournament Topics (Regional & State topics + the following) Components of a gene
Sanger sequencing Random vs. targeted mutagenesis Mechanism of DNA replication, including roles of enzymes DNA fingerprinting and RFLP analysis Post-transcriptional RNA processing and regulation Mechanism of gene expression, including roles of enzymes Phylogenetics
RNA-Seq, Tn-Seq, and their uses Promoter structure DNA microarrays DNA repair Molecular consequences of mutations Plasmid cloning, selection, and isolation Epigenetics Organelle DNA
Gene therapy, CRISPR-Cas technology Comparison of Next Generation Sequencing Platforms CENTRAL DOGMA OF MOLECULAR GENETICS DNA ---- RNA --- PROTEIN SYNTHESIS REPLICATION TRANSCRIPTION TRANSLATION Exceptions among viruses RNA to DNA (retroviruses) DNA Structure
Replication (in nucleus) DNA uncoils & splits Reads 3 to 5 Assembles 5 to 3 4 types of nucleotides Okazaki fragments in lagging strand Prokaryotic vs. Eukaryotic PROKARYOTIC REPLICATION
Eukaryotic replication ENZYMES FOR REPLICATION DNA Repair Genes encode proteins that correct mistakes in DNA caused by incorrect copying during replication and environmental factors such as by-products of metabolism, exposure to
ultraviolet light or mutagens The DNA repair process must operate constantly to correct any damage to the DNA as soon Gene Expression Transcription DNA is template for making RNA (in nucleus) Translation (protein synthesis) - in cytoplasm at the ribosome. m-RNA has blueprint, t-RNA transfers amino acids, and Ribosome (r-RNA) allows T-RNA to attach to M-RNA at
appropriate site Transcription Making RNA from DNA template Transcription takes place in the nucleus Types of RNA Differences between RNA & DNA
RNA is single strand - DNA is double strand RNA has Ribose DNA has Deoxyribose RNA has Uracil DNA has Thymine Messenger RNA carries blueprint from nucleus to cytoplasm acts as template Transfer RNA brings amino acids Ribosomal RNA part of ribosome Polycistronic mRNA vs.
Monocistronic mRNA Polycistronic mRna contains for more than one cistron codes for fore than one protein is transcribed from more than one gene (cistron) and has as many initiation and termination codes is present in prokaryotes Monocistronic mRNA
contains codons of a single cistron codes for a single protein is transcribed from a single gene (cistron) and has one initiation and termination codon is present in eukaryotes Promoters
region of DNA that initiates transcription of a particular gene located near the genes they transcribe, on the same strand and upstream on the DNA (towards the 3' region of the anti-sense strand also called template strand Prokaryotic Gene Organization Prokaryotic Promoter
Eukaryotic Gene Organization Eukaryotic promoter RNA Processing mRNA in prokaryotic cells is to function after transcription but in eukaryotic cells it is modified after transcription RNA Processing includes 5 capping
for RNA stabilization and ribosome binding; splicing for removing intron sequence and 3 polyadenylation for protecting mRNA from 3 exonuclease, extending the half life of mRNA Eukaryotic pre-mRNA is converted Post-transcription Modifications
Introns and exons at transcription Introns removed Exons are coding pieces for protein synthesis Cap and PolyA tail are added Universal Genetic Code
Special start codon (AUG) and three stop codons (UAA, UAG and UGA) Many codons may code for same amino acid Third position of the codon, it is more likely the nucleotide is different but it still may code for same amino acid (wobble) Universal Code (Codon = Amino Acid) Translation (Protein
Synthesis) The steps of translation: Eukaryotic 1. Initiation: mRNA enters the cytoplasm and becomes associated with ribosomes (rRNA + proteins). tRNAs, each carrying a specific amino acid, pair up with the mRNA codons inside the ribosomes. Base pairing (A-U, G-C) between mRNA codons and tRNA anticodons determines the order of amino acids in a protein. 2. Elongation: addition of amino acids one-by-one: As the ribosome moves along the mRNA, each tRNA transfers its amino acid to the growing protein chain, producing the protein 3. Termination: when the ribosomes hits a stop codon UAA, UGA, or UAG - the ribosome falls apart Note: The same mRNA may be used hundreds of times during translation by many ribosomes before it is degraded (broken down) by the cell
Control of Gene Expression in Prokaryotes Important for single celled organisms who depend on environment for all activities Bacteria use operons - many functional-related genes are clustered and transcribed under the same types of
regulation Lac & Trp Operons - examples of prokaryotic gene regulation Lac Operon The genes that code for the enzymes needed for lactose catabolism are clustered on the same chromosome in what is called the Lac Operon The E. coli only express the genes and make these enzymes when lactose is available to be metabolized. This is an inducible operon where
genes are expressed in the presence of a substance Trp Operon The genes for the five enzymes in the Trp synthesis pathway are clustered on the same chromosome in what is called the Trp Operon This is a repressable operon where the
operon are turned off in the presence of a substance Regulatory Components in Eukaryotes Enhancers - short regions of DNA that can be bound with proteins to promote expression of a distal or a proximal gene. Promoters - proximal DNA
sequences that binds to RNA polymerase for regulating gene expression. TATA Box - binds to transcription factor for regulating gene expression, usually within 30bp of the transcription start site. Expression in Eukaryotes
Transcriptional Control Post transcriptional Control assembling proteins Cell differentiation and specialization Turning genes on and off Chemical Signals Hormones Chemical Modifications Relocation of DNA transposons Nuclear vs Cytoplasmic DNA in Eukaryotic Cells
Nuclear DNA in chromosomes within the nucleus of the cell Cytoplasmic or Organelle DNA in chloroplasts and mitochondria Chloroplast DNA (cpDNA) Mitochondrial DNA (mtDNA) Features: Maternal inheritance Resemble prokaryotic DNA Slow accumulation of mutations Organelle DNA
Mitochondria and Chloroplasts have DNA similar to Prokaryotic cells It is believed that these organelles were once independent prokaryotes who took up residence and formed a mutualistic relationship They are involved in energy MITOCHONDRIAL INHERITANCE
The inheritance of a trait encoded in the mitochondrial genome Mitochondrial DNA or mtDNA is inherited from the mother The mtDNA is circular and resembles prokaryotic DNA The mitochondria are responsible for energy
production cellular respiration Mutations Mutation any change in the DNA blueprint for making protein or RNA Gene mutation Chromosomal mutation Agents causing mutations radiation, chemicals, excess
heat Genetic Disorders Nondisjunction extra or missing chromosomes as Downs Syndrome Trinucleotide repeats triplet nucleotides repeated too often as Huntingtons Defective genes does not produce
correct protein as sickle cell anemia (A & T traded places) Human genetic disorders can be dominant, recessive, sex-linked, epistatic, variable expressed Biotechnology
Technology used to manipulate DNA Procedures often called genetic engineering Recombinant DNA - DNA from two sources Transgenic individuals have DNA from another organism Often involve putting genes into viruses or bacteria. Vectors are the pieces of DNA used to transfer genes into a host cell often Overview of Biotechnolo gy
identifying recognizing desired DNA fragment or plasmid using radioactive probes cutting DNA - using desired restriction enzymes or enzymatic sissors making hybrids of DNA using Hybridization techniques Storing, analyzing and using clones in biotechnology processes
Basic Tools of DNA Technology Identifying desired DNA Cutting DNA with Restriction Enzymes
Inserting DNA into Vector as Plasmid Connecting DNA pieces with Ligase Inserting Vector into Host Cell as bacterium Cloning desired DNA and Vectors Storing clones in DNA Libraries Identifying cloned genes with Radioactive Probes Analyzing DNA by cutting fragments and separating by Electrophoresis Techniques
Cloning within cells and with PCR Storing clones in DNA Libraries Identifying cloned genes with Radioactive Probes Analyzing DNA by cutting fragments and separating by Electrophoresis/nucleic acid hybridization/DNA probes Transferring DNA from gel by Blotting Imaging with autoradiography DNA Sequencing to determine exact
sequence Microassays to analyze gene function Cloning Methods Currently in Use Traditional Restriction digestion cloning o Plasmids and inserts are digested with the same restriction enzymes and then ligated together. Gateway Recombination
o Regions of homology between insert DNA and plasmid are used for a recombination event that transfers the insert DNA into the plasmid. Gibson assembly o DNA fragments containing homologous overlapping ends are ligated together in one reaction. TA cloning o Linearized plasmids engineered to have single T overhangs are ligated together with a PCR product insert. Most DNA polymerases leave an A overhang on PCR products, which allows them to base pair with the T overhangs on the TA cloning plasmid. Cloning into A Plasmid
Gene selection Plasmid selection Putting pieces together Insert into host bacteria Gene Selection and cDNA complementary DNA
Eukaryotic genes contain introns but bacteria do not contain the necessary enzymes to remove introns Eukaryotic genes that are inserted into bacteria must be inserted without introns. Use reverse transcriptase (from retroviruses) and modified MRNA to produce cDNA with introns already removed
Plasmid Selection and Isolation A small DNA molecule that is physically separate from, and can replicate independently of, chromosomal DNA within a cell as a bacterium When used in genetic engineering called vectors Several methods to isolate
plasmid DNA from bacteria Molecular Cloning To isolate a piece of DNA and amplify it via recombinant DNA technology. The DNA source can be genomic DNA, cDNA, or PCR amplified DNA fragments. These DNA pieces are cut by restriction enzymes to create
compatible DNA ends with the vectors. Polymerase Chain Reaction (PCR) Technique for quickly making an unlimited number of copies of any piece of DNA
Sometimes called "molecular photocopying DNA Libraries Genomic normal DNA cDNA modified to remove introns
Fragments stored Stored in plasmids or bacteriophages Hybridizatio n Base pairing of two single strands of DNA
or RNA. Can be DNADNA, DNA-RNA Can be a radioactive probe Radioactive Probes short, radioactive strands of DNA will pair up with complementing strands of DNA fragments that contain the labeled
pieces will show up on an x-ray film Electrophoresis A process in which molecules (such as proteins, DNA, or RNA fragments) can be separated according to size and electrical charge by applying an electric current to them. DNA Analysis DNA Sequencin g
Determine the exact nucleotide sequence Columns for A,T,C, and G Read rows from top to bottom sequenced strand is 5' to 3' ACGCCCGAGTAGCCCAGA
TT DNA Fingerprinting Techniques RFLP Restriction Fragment Length Polymorphism (original) PCR Polymerase Chain Reaction VNTRs Variable Number Tandem
Repeats STRs - Short Tandem Repeats Ribosomal DNA analysis Y-chromosome analysis Restriction Fragment Length Polymorphism RFLP The DNA of an organism is cut up into fragments using restriction enzymes.
A large number of short fragments of DNA will be produced (RFLPs) Electrophoresis is a technique used to separate the DNA fragments according to their size Uses- identification of diseased genes including oncogenes, identification of viral infections, determining family relationships among individuals, and identifying tissue found at a crime scene Restriction Fragment Length Polymorphisms (RFLP)
Genetic variations at the site where a restriction enzyme cuts a piece of DNA Such variations affect the size of the resulting fragments These sequences can be used as markers on physical maps and linkage
VARIABLE NUMBER TANDAM REPEATS (VNTRs) Short nucleotide sequences Organized in clusters of tandem repeats VNTR = 14-100 base pairs SNR = 2- 10 base pairs Restrictio n Mapping Description of
restriction enzyme cleavage sites within a piece of DNA Use of different restriction enzymes to analyze and generate a physical map of genomes, genes, or other segments of DNA DNA Microassay
studying how large numbers of genes interact with each other precisely apply tiny droplets containing functional DNA to glass slides attach fluorescent labels to DNA from the cell they are studying. labeled probes are allowed to
bind to complementary DNA strands on the slides slides are put into a scanning microscope that can measure the brightness of each fluorescent dot RNA-Seq RNA-seq refers to the method of using Next Generation Sequencing (NGS) technology to measure a set of RNA levels
NGS technology is an ultra-high-throughput technology to measure DNA sequences Advantages of RNA-seq over microarray include: 1) Wider measurable range of expression levels 2) Not dependent on known genome 3) Free of hybridization artifacts 4) Possibility of one platform for all Next Generation Sequencing Platforms Applications of Biotechnology Techniques
Human Genome Project Diagnosis of Disease PCR & DNA probes Human Gene Therapy Vaccines & Pharmaceutical Products Forensics DNA Fingerprints (RFLP & VNTR) Environmental Recycling & detoxification Agricultural transgenic organisms
gene therapy genetic testing bioterrorism Epigenetics The study of heritable changes in gene activity that occur without a change in the sequence of the genetic material. Epigenetics literally means in addition to genetics. Epigenetic factors can regulate the amount of gene activity, influencing the growth and
appearance of an organism Malfunctions in epigenetic control of gene activity have been implicated in cancer, cardiovascular disease and several inherited genetic conditions Phylogeneti cs Study of evolutionary relationships among groups of organisms based upon their genetics o Relationships between organisms are often represented using phylogenetic trees. o Organisms more closely related to each other are
physically closer on the tree. o Common ancestors are represented by nodes that connect branches together. Has taxonomy folks in a turmoil they cant agree so we have national lists for our taxonomy events
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