2013 DESIGNER GENES TRAINING GUIDE by Karen L.

Lancour
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. BE SURE TO CHECK THE 2013 EVENT RULES for EVENT PARAMETERS and TOPICS FOR EACH COMPETITION LEVEL TRAINING MATERIALS: Training Power Point presents an overview of material in the training handout Training Handout presents introductory topic content information for the event Sample Tournament has sample problems with key Event Supervisor Guide has event preparation tips, setup needs and scoring tips Internet Resource & Training Materials are available on the Science Olympiad website at www.soinc.org under Event Information. A Biology-Earth Science CD as well as the Division B and Division C Test Packets are available from SO store at www.soinc.org Students will solve problems using their knowledge of Molecular Genetics, Biotechnology, and Population Genetics. This event may be run as stations but it need not be. It is a very different event when run as paper pencil. The best competition is still as stations using process skills and problem solving. Every attempt should be made to avoid over-emphasis on a particular area. At the various levels, areas to be tested include:
Regional & State DNA structure & function DNA Semi-conservative Replication Gene expression (transcription and translation Control and detection of gene expression Mutations DNA Sequencing Regional & State Lac & Trp Operons DNA Fingerprinting/RFLP DNA Sequencing Plasmid selection and isolation PCR Gene Therapy National (all topics) Restriction mapping Mitochondrial DNA DNA Repair Post- transcriptional modification Trinucleotide repeats Epigenetics

Note: Regions or States may decide to cover all of the topics so check with your local tournament director for specifics.

Process skills may include observations, inferences, predictions, data analysis, and calculations. Note: It is a good idea to review the General Genetics Prinicples that are in the Heredity Event in Division B they are very relavent to Designer Genes covers.

MOLECULAR GENETICS

CENTRAL DOGMA OF MOLECULAR GENETICS

DNA ---- RNA --- PROTEIN SYNTHESIS REPLICATION TRANSCRIPTION TRANSLATION Central dogma of molecular genetics is DNA - RNA - Protein. Exception is in retroviruses where genetic storage vehicle is RNA. It then makes a DNA which replicates to form double stranded DNA and continues through dogma. Exceptions among viruses RNA to DNA (retroviruses)

DNA Structure
DNA structure double helix with sugar (deoxyribose), phosphate and nitrogen bases (Adenine, Thymine, Guanine, and Cytosine) Pairing A with T and G with C

Nucleotide - basic unit of sugar, phosphate and nitrogen base - 4 kinds of nucleotides because of the types of bases

DNA Replication
DNA replication is semi-conservative and occurs in the nucleus.

Events that occur: DNA polymerase is the key enzyme DNA uncoils and splits template strand is read 3 to 5 new complementary strand must add new nucleotides to the 3 end leading strand (continuous) while lagging strand is fragments (Okazaki fragments) latter attached with enzyme ligase

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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 as it occurs.

ENZYMES INVOLVED IN REPLICATION

The replication fork is the unwound helix, with each strand being synthesized into a new double helix

Topoisomerase is responsible for initiation of the unwinding of the DNA. Helicase accomplishes unwinding of the original double strand, once supercoiling has been eliminated by the topoisomerase. DNA polymerase (III) proceeds along a single-stranded molecule of DNA, recruiting free dNTP's (deoxy-nucleotide-triphosphates) to hydrogen bond with their appropriate complementary dNTP on the single strand (A with T and G with C), and to form a covalent phosphodiester bond with the previous nucleotide of the same strand. DNA polymerases cannot start synthesizing de novo on a bare single strand. It needs a primer with a 3'OH group onto which it can attach a dNTP. DNA polymerase also has proofreading activities, so that it can make sure that it inserted the right base, and nuclease (excision of nucleotides) activities so that it can cut away any mistakes it might have made. Primase attaches a small RNA primer to the single-stranded DNA to act as a substitute 3'OH for DNA polymerase to begin synthesizing from. This RNA primer is eventually removed and the gap is filled in by DNA polymerase (I). Ligase can catalyze the formation of a phosphodiester bond given an unattached but adjacent 3'OH and 5'phosphate. This can fill in the unattached gap left when the RNA primer is removed and filled in. Single-stranded binding proteins are important to maintain the stability of the replication fork. Single-stranded DNA is very labile, or unstable, so these proteins bind to it while it remains single stranded and keep it from being degraded.

Nuclear vs Cytoplasmic DNA in Eukaryotic Cells

Nuclear DNA in chromosomes within the nucleus of the cell Cytoplasmic DNA in chloroplasts and mitochondria Chloroplast DNA (cpDNA) Mitochondrial DNA (mtDNA) Features: Maternal inheritance Resemble prokaryotic DNA Slow accumulation of mutations

Differences between RNA & DNA

RNA is single strand - DNA is double strand RNA has Ribose DNA has Deoxyribose RNA has Uracil DNA has Thymine

Gene Expression
There are two processes with utilize the DNA template code to ultimately produce proteins: Transcription DNA is template for making RNA (in nucleus) There are 3 types of RNA. 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. many factors control gene expression including: o factors affecting DNA structure, o gene expression, o factors affecting assembly of proteins after o translation, o hormones, o environmental factors as viruses.

Types of RNA
Kinds of RNA three kinds of RNA are produced in the nucleus using DNA coding strands

Messenger RNA (m-RNA) carries genetic code from DNA into cytoplasm Transfer RNA (t-RNA) brings the amino acids for building of protein to be attached according to the genetic code of the M-RNA Ribosomal RNA (r-RNA) make up the ribosome and reads the code of M-RNA and allow TRNA to attach and connect amino acids

Transcription
Transcription - production of RNA in the nucleus using a DNA segment as a template and RNA polymerase as the key enzyme.

Post-transcription Modifications
RNAs are modified in eukaryotes before leaving the nucleus.

PreM-RNA has exons (coding segments) and introns (noncoding segments between exons) introns (the noncoding segments) are removed a cap is added to the 5 end a poly A tail is added to the 3 end before it leaves the nucleus

Universal Code (Codon = Amino Acid)

Each three base codon on the messenger RNA (m-RNA) is a code for an amino acid There are 64 possible three base codons 61 are codes for one of the 20 amino acids The three remaining codons are termed stop codons because the signal the end of a peptide segment Notice that many of the amino acids have more than one codon A three base code on the DNA produces the mRNA codon The three base code on the t RNA is termed an anticodon because it will bond to a m-RNA codon during translation or protein synthesis

Translation (Protein Synthesis)

Translation genetic code used to form amino acid sequence using M-RNA, T-RNA, and R-RNA (ribosomes) occurs in the cytoplasm at the ribosome. Many key enzymes (proteins) are involved.

Translation (Protein Synthesis) The steps of translation:

Initiation: a mRNA enters the cytoplasm and becomes associated with ribosomes (rRNA + proteins) and tRNAs, each carrying a specific amino acid, pair up with the mRNA codons inside the ribosomes. The base pairing (A-U, G-C) between mRNA codons and tRNA anticodons determines the order of amino acids in a protein. Elongation: involves the 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 Termination: when the ribosomes hits a stop codon - UAA, UGA, or UAG - no tRNA with its amino acid can be added so the ribosome falls apart and the process ends. The same mRNA may be used hundreds of times during translation by many ribosomes before it is degraded (broken down) by the cell.

A close up showing the M-RNA (with its codon) and T-RNA (with it anticodon as well as the Amino Acid) attaching at the P and A sites on the Ribosome.

Control of Gene Expression

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 Abnormal Expression of Genes

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Mutations
Gene section of DNA with carries the blueprint for making a peptide strand or RNA. Mutation changes in genetic code (DNA blueprint) of genes or chromosomes and causes changes in expression in the for making protein or RNA Gene mutation Chromosomal mutation Agents causing mutations radiation, chemicals, excess heat , viruses

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) Causes of mutations chemicals, radiation, temperature, viruses Genetic disorders and their causes as nondisjunction (Downs syndrome), trinucleotide repeats (fragile X and Huntingtons), defective genes (sickle cell anemia, hemophilia) Trinucleotide repeats sequences of 3 nucleotides is repeated, often several times in a gene, when too many repeats are formed cause genetic disorders Nondisjunction chromatids do not separate properly during meiosis. Individual formed from such gametes have extra or missing chromosomes. Human genetic disorders can be dominant, recessive, sex-linked, epistatic, variable expressed Crossover frequency during meiosis, pieces trade places determining frequency

Mitochondrial Inheritance genetic make-up of mitochondria, genetic code and patterns

transmitted through mother. 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 Mitochondria can reproduce independent of the rest of the cell an advantage in energy production

Persons with a mitochondrial disease may be male or female but they are always related in the maternal line and no male with the disease can transmit it to his children

Mitochondrial myopathies are a group of neuromuscular diseases caused by damage to the mitochondria-small, energy-producing structures that serve as the cells' "power plants."

Lac & Trp Operons - examples of prokaryotic gene regulation

Many of the prokaryotic genes as in E.coli are expressed or are always turned "on".
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Others are active only when their products are needed by the cell, so their expression must be regulated. Examples of Operons in E. coli o The genes for the five enzymes in the Trp synthesis pathway are clustered on the same chromosome in what is called the Trp Operon - If the amino acid tryptophan (Trp) is added to a culture of E coli , the bacteria soon stop producing the five enzymes needed to synthesize Trp from intermediates produced during the respiration of glucos so the presence of the products of enzyme action represses enzyme synthesis This is a repressable operon where genes are expressed in the absense of a substance and the presense of the substance shuts off the gene

The genes that code for the enzymes needed for lactose catabolism are clustered on the same chromosome in what is called the Lac Operon prokaryotics as E. coli have a mechanism for metabolizing lactose the sugar used for energy. Three proteins or enzymes are needed in lactose metabolism and they are encoded in a single expressible unit of DNA 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
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Biotechnology technology to manipulate DNA techniques often called genetic engineering or

Recombinant DNA Technology. 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 plasmids of bacteria

Overview of Biotechnology

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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

DNA Analysis Technologies 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 cloning DNA using other cells or in a test tube as with PCR Polymerase Chain Reaction clones DNA segments in a test tube quickly and inexpensively. May use very small amounts of DNA to clone storing DNA in DNA libraries of plasmids or bacteriophages of genome DNA or cDNA. separating DNA segments with electrophosesis transferring DNA using blotting imaging DNA with autoradiography analyzing DNA by sequencing or determining the nucleotide sequence of a gene, microassays analyze gene function and expression, DNA fingerprinting techniques as RFLP or restriction fragment length polymorphism, VNTRs or Variable Number Tandem Repeats, STRs or Short Tandam Repeats, Ribosomal DNA Analysis, or Y-chromosome Analysis

Basic Terminology

Recombinant DNA DNA from two different sources combined. Often involve putting genes into viruses or bacteria using a vector. Inserting a gene into a bacterium - Organism provides the desired piece of DNA which is spliced into a piece of DNA used to transfer the genes or vector which is inserted to a Host cell (often a bacterium) Plasmids in bacteria, circular DNA serve as vectors. Easily taken up by bacterial cells. It is more difficult to insert vector into Eukaryotic cells. Transgenic organisms have DNA from another organism Restriction enzymes - enzymes to cut DNA at a particular spot and DNA ligase enzymes reattach ends. Hybridization process of putting pieces of DNA together. Chromosome mapping determining the location of genes on a chromosome and making a map of restriction sites as Retriction Maps.

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Basic Tools
Gene selection & isolation from Donor Vector - Plasmid selection & isolation Restriction enzyme to cut piece Putting pieces together (hybridization) DNA ligase to reattach pieces Insert into Host bacteria Clone the bacteria

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cDNA complementary DNA

cDNA- 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 M-RNA to produce cDNA with introns already removed

Techniques
Storing clones in DNA Libraries Cloning within cells and with PCR 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

DNA Libraries

Genomic normal DNA cDNA modified to remove introns Fragments stored Stored in plasmids or bacteriophages

Polymerase Chain Reaction (PCR)

Technique for quickly making an unlimited number of copies of any piece of DNA
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Sometimes called "molecular photocopying

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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 Hybridization
Base pairing of two single strands of DNA or RNA. Can be DNA-DNA, DNA-RNA Can be a radioactive probe
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DNA Analysis

DNA Sequencing
Determine the exact nucleotide sequence Columns for A,T,C, and G Read rows from top to bottom

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Restriction 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 brightness reveals how much of a specific DNA fragment is present, an indicator of how active it is.

DNA Fingerprinting Techniques RFLP Restriction Fragment Length Polymorphism (original) PCR Polymerase Chain Reaction
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 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. 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 maps.

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VARIABLE NUMBER TANDAM REPEATS (VNTRs)

Short nucleotidesequences

Organized in clustersof tandem repeats

VNTR = 14-100 base pairs SNR = 2- 10 base pairs

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Applications of Biotechnology Techniques

Human Genome Project - entire gene make up of humans 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 - changing the expression of a persons genes - body (somatic) or germ cells
done invitro or exvitro.

Bioethics
Major concerns concerning safety and ethics of recombinant DNA technology.

Potential Hazards vs. Potential Gains Concerns: o genetically modified foods o genetically engineering microbes o cloning whole organisms o embryonic stem cell research o gene therapy o genetic testing o bioterrorism

Epigenetics is 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 There are several epigenetic ways in which gene activity can be prevented or controlled, including modification of histone proteins DNA methylation RNA interference For any of these methods of gene regulation, the absence of the protein product of the gene causes a change in the function or development of the cell malfunctions in epigenetic control of gene activity have been implicated in cancer, cardiovascular disease and several inherited genetic conditions

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SAMPLE STATION PROBLEMS

DNA structure and Replication

Production of RNA from a coding strand of DNA - & post transcription modification of M-RNA Protein Synthesis Analysis (Replication and Transcription)

Control and Detection of Gene Expression Chromosome map units problems Cross over frequencies

DNA sequencing Restriction enzyme analysis Making a restriction map of a plasmid or DNA segment. Interpreting data from DNA fingerprinting studies

Forensic Uses of Biotechnology

Interpreting DNA analysis data + comparing RFLP and PCR forensic testing and analysis Analyzing blood chemistry, blood typing and blood cell genetic information Analyzing and interpreting chromosome maps Understanding and interpreting the uses of restriction enzymes and radioactive probes

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