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Biotechnology and Genomics _________________ is the production of identical copies of DNA, cells or organisms If you think about it, members of a bacterial colony in a sample are clones because they all came from division of the same cell ____________ twins are even clones Single embryo separate to become two. _______ ______ is production of many identical copies of the same gene. If the inserted gene is replicated and expressed, we can recover the _______________or ________________. Cell containing gene of interest Bacterium 1 Gene inserted into plasmid How It’s Done Bacterial chromosome Plasmid Recombinant DNA (plasmid) Gene of interest 2 Plasmid put into bacterial cell Recombinant bacterium DNA of chromosome • • • • Cuts DNA at specific points. Like… Cleaves vector (plasmid) and foreign (human) DNA. Cleaving DNA makes DNA fragments ending in short single-stranded segments with “______________” These allow for insertion of foreign DNA into vector DNA. A G T C A G A A T T C G C T C T T A A G C G A A T T T T A Google: McGraw-Hill “Restriction Endonucleases” C G C G C G A 5 DNA Ligase Remember its function in DNA replication? • Job? • DNA “_____________” has now occurred • The cell membranes of bacteria can be made more permeable to take up plasmids at this point. – Bacteria and plasmids will reproduce. – Many copies of the plasmid and many copies of the foreign gene Google: mcgraw-hill: Go to www.sumanasinc.com are now produced. “Steps in Cloning a Animations-molecular biology-plasmid cloning gene” 6 DNA Cloning: Polymerase Chain Reaction (____) • Amplifies a targeted sequence of DNA – Create millions of copies of a single gene or a specific piece of DNA in a test tube • Requires: – 1. ______ __________ from hot-springs resident bacterium, Thermus aquaticus (called ______ _____________) • Withstands the temperature necessary to separate double-stranded DNA. – 2. A supply of __________________________ for the new, complementary strand – 3. _________________: ~10-20 base sequences which match the ends of the DNA sequence of interest 7 PCR A three-step cycle—______________, ________________, ____________ —brings about a chain reaction that produces an exponentially growing population of identical DNA molecules PCR cycles DNA copies first 1 second 2 third 4 fourth 8 fifth 16 DNA double strand new strand new old strand old old new and so forth Go to www.sumanasinc.com Animations-molecular biology-PCR 8 5 TECHNIQUE 3 Target sequence 3 Genomic DNA 1 5 5 3 3 5 2 Cycle 1 yields 2 molecules 3 Advertisement for thermocycler (machine used for PCR) http://www.cnpg.c om/video/flatfiles/ 539/ Cycle 2 yields 4 molecules Cycle 3 yields 8 molecules; 2 molecules (in white boxes) match target sequence Applications of PCR: Analyzing DNA Segments • ________ ________________________ is the technique of using DNA fragment lengths called MCGRAW RFLP’s to match/investigate DNA samples. GOOGLE: HILL “RESTRICTION FRAGMENT LENGTH • HOW? POLYMORPHISM” – By treating DNA samples with restriction enzymes, a unique collection of different length fragments is produced – Electrophoresing these fragments separates the fragments according to their • __________________ – Usually used to measure number of repeats of short sequences – Produces distinctive banding pattern 10 What is Electrophoresis? Electrophoresis is a laboratory technique for separating mixtures of charged molecules. • __________________: a material composed of two or more elements or parts. •___________________: a molecule (such as a protein or DNA) that has too many or too few electrons. Charged molecules are separated based on their electrical charge and size. Mixture of Charged Molecules Essentials parts of GEL ELECTROPHORESIS • ___________________________– the flow of electric charge • Acts as a _____________, a device to filter small particles out of a mixture of larger particles. How Separation Occurs Many molecules (amino acids, peptides, proteins, DNA, and RNA) have naturally occurring negative and positive charges on them. The sum of these charges determines the overall charge. When introduced to an electrical current, negatively charged molecules are attracted to the positive electrode and positively charged molecules are attracted to the negative electrode. N+ O N Positively Charged Amino Acid - - + + - + + Positively Charged Peptide + - + - + + + - -+ + Negatively Charged Protein How Separation Occurs Molecule Size: The porous material is made of microscopic particles suspended in a gel. The microscopic particles attach to one another forming tunnels that act as a sieve to separate the molecules. Small molecules can move faster than large molecules. Porous Material Fig. 20-9a TECHNIQUE Mixture of DNA molecules of different sizes Power source Anode – Cathode + Gel 1 Power source – 2 + Homework: Do a virtual DNA fingerprinting exercise GOOGLE: NOVA + CREATE A DNA FINGERPRINT Restriction fragment analysis is useful for comparing two different DNA molecules, such as two alleles for a gene Normal -globin allele 175 bp DdeI Sickle-cell allele Large fragment 201 bp DdeI Normal allele DdeI DdeI Large fragment Sickle-cell mutant -globin allele 376 bp DdeI 201 bp 175 bp Large fragment 376 bp DdeI DdeI restriction sites in normal and sickle-cell alleles of -globin gene DdeI Electrophoresis of restriction fragments from normal and sickle-cell alleles DNA Fingerprinting & Paternity Mother Child Male 1 few Male 2 Base repeat units Who is the father? Male 1 or 2? many DNA Band patterns Fluorescence units a. Increasing size GOOGLE: MCGRAW-HILL “DNA fingerprinting” b. Automated DNA fingerprinting Go to www.sumanasinc.com 20 Animations-biotechnology-paternity testing STR’s: short tandem repeats A method of profiling called STR analysis uses loci in DNA where the same sequences of bases may occur a number of times. For example the sequence GACT may be repeated 5x in one individual and 20x in another individual. The number of repeats varies from person to person This can help identify individuals from each other or biological evidence This is the FBI’s 13 Loci standard for matching individuals to evidence. The possibility of two individual’s coding the same in STR’s for the 13 loci 21 is greater than 1 in a billion Biotechnology Products • Genetically engineered organisms can produce biotechnology products. They are called “genetically modified organisms” or (_______’s) • Organisms that have had a foreign gene inserted into them are ______________. 22 Transgenic Bacteria • Genes of interest are inserted into bacteria. • Bacteria are grown in large vats called ________________ and product is harvested. – Products on the market include insulin, hepatitis B vaccine, tPA, and human growth hormone. • Some genetic modifications can promote the health of plants and other can degrade harmful 23 Transgenic Plants • Agricultural Crops – Foreign genes now give cotton, corn, and potato strains the ability to produce an insect toxin – Soybeans are now resistant to a common herbicide – Weed-killer resisting crops are now common (“Roundup” ready) Human Hormone Production: 24 Transgenic Animals • Vortex Mixing: – Many types of animal eggs have taken up the gene for bovine growth hormone (bGH) through a procedure that uses agitation, tiny needles and foreign DNA to insert a gene into the egg. – The procedure has been used to produce larger livestock animals 25 Gene Pharming: microinjection of human gene -Use of transgenic farm animals to produce pharmaceuticals . Genes coding for therapeutic & diagnostic proteins are incorporated into an animal’s DNA -The proteins appear in the animal’s milk . Plans are to produce drugs to treat: 26 Treating Disorders ◦ __________ ______ involves procedures to give patients healthy genes to make up for a faulty gene. It also includes the use of genes to treat genetic disorders and various human illnesses. There are __________ (outside body) and Ex Vivo __________ (inside body) of ◦ Inmethods Vivo Children with Severe Combined gene therapy. Immunodeficiency (SCID) 27 Human Genome Project _________________ is the study of genomes of humans and other organisms. _____________- All the genetic information of an individual (or species) Goals of Human Genome Project Did it ever get done? How many base pairs? 29 Findings of the Human Genome Project • Humans have between ____________ genes (still being researched) – Most genes code for proteins – Much of the other DNA was formerly described as “junk” • “Junk” did not specify the order of amino acids in a polypeptide Recent observations suggest that between 74% and 93% of the genome is transcribed into RNA but not all translated into proteins Therefore, the “junk” (nontranslated RNA) may code for regulatory gene expression necessary for proper cellular events. 30 Gene Structure Historically, genes were defined as discrete units of heredity that corresponded to a locus on a chromosome. Prokaryotes typically possess a single circular chromosome without ____________ and have less DNA. Eukaryotic chromosomes with much more DNA are much more complex. 31 Eukaryotic Gene Structure • ___________________________ are DNA sequences that occur between genes • __________________ DNA elements occur when the same sequence of two or more nucleotides are repeated many times along the length of one or more chromosomes. Remember STR’s?... Can also be referred to as ______________ • __________________ are specific DNA sequences that have the remarkable ability to move within and between chromosomes. AKA: jumping genes! YouTube: Transposons: Shifting segments of the genome 32 • The study of the structure, function, and interaction of cellular proteins • At least 20-25K of our genes are translated into proteins • The sum total of these is called the human __________ • Understanding protein function is essential to the development of better drugs. How? • Once the primary structure of these protein is known – It should be possible to predict their tertiary structure – Computer modeling of the tertiary of these proteins is an important part of proteomics Worth a look -one last link for biotech products from BIO-RAD: http://bio-rad.cnpg.com/Video/flatFiles/799/ 33 Current understanding of what a “Gene” is… • A genomic sequence (either DNA or RNA) directly encoding functional products, either RNA or protein. 34