BIOL 4003 Week 12 Notes
BIOL 4003 Week 12 Notes 4003
U of M
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This 8 page Class Notes was uploaded by Rachel Heuer on Sunday April 17, 2016. The Class Notes belongs to 4003 at University of Minnesota taught by Robert Brooker in Spring 2016. Since its upload, it has received 9 views. For similar materials see Principles of Genetics in Biology at University of Minnesota.
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Date Created: 04/17/16
Chapter 20: - Recombinant DNA: use of in vitro molecular techniques to isolate/manipulate fragments of DNA o Isolating and making many copies of new DNA molecules is gene cloning § Often uses PCR and amplifies gene frequency o Recombinant DNA technology has led to an increase in knowledge of DNA mechanisms, structure, and function - Cloning experiments: involve two kinds of DNA molecules o Need chromosomal DNA (source of DNA segment of interest) and vector DNA § Vector can replicate independently of chromosomal DNA and serves as the carrier so that it may be cloned o to prepare chromosomal DNA § Obtain tissue from organism of interest § Break cells § Extract and purify host DNA o Needs restriction endonucleases § Insertion of chromosomal DNA into a vector requires the cutting and joining of DNA fragments • Enzymes used to cut DNA are known as restriction endonucleases or restriction enzymes § Recognizes and binds to specific DNA sequences and then cleave the DNA at two defined locations, one point on each strand § DNA ligase links backbones back together when new DNA comes in § Can create “sticky ends” • Can bond but is not very stable because there are only a few hydrogen bonds • Ligase is added, which connects them to make the connection stable by forming covalent bonds (making the bond more permanent) o B-globin example § Trying to clone human b-globin gene into plasmid vector § Vector contains two important genes already • ampR o antibiotic resistance o identifies cells that have TAKEN UP the vector • LacZ: encodes b-galactosidase o Identifies which bacteria have picked up the cloned gene § Cut vector and normal DNA with the same restriction enzyme to create sticky ends (vector only contains one restriction site for that specific restriction enzyme) • When coming back together there are 3 possible hybrid vectors o Plasmid + gene of interest o Original plasmid (strands come back together) o Plasmid and another piece of chromosomal DNA (but NOT gene of interest) § Expose mixture of recombinant DNAs to bacterial cells that do not have a plasmid • Bacterial cells will take up the plasmid § Plate with ampicillin and Xgal • Cells need vector (contains ampR) to grow o Antibiotic resistance • We don’t want blue colonies (we want white because our gene of interest is in the middle of lacZ gene) o When gene breaks up lacZ gene, b-galactosidase cannot be made and colonies are white o THAT IS WHAT WE WANT • Cells that take up a vector/DNA are “competent” o Transformation: when plasmid vectors are used o Transfection: when a viral vector is introduced into a host cell § During transformation, a single bacterial cell usually takes up a single copy of the vector § Amplification of a cloned gene occurs in two ways: • Host cell replicates vector many times o This generates a lot of copies per cell • Bacterial cell divides quickly o Generates a population of many cells § Cell typically only picks up one copy (but gets replicated) - Recombinant DNA can be used for stuff other than cloning genes - cDNA: clones DNA starting with sample of RNA o uses reverse transcriptase to make a complementary strand of DNA off of RNA template o used by retroviruses to copy their RNA genome to DNA o DNA that is made from RNA is called complementary DNA (cDNA) § Can be double or single stranded o DNA primer with many T’s must be added so that it can bind to the poly-A tail of the RNA and start reverse transcription § Can then chop up RNA template strand to make RNA primers for DNA polymerase to fill in the gaps with DNA and get rid of the RNA primers, making the DNA double stranded § DNA ligase is added to make the complementary strand continuous - PCR: Polymerase Chain Reaction o Way to copy DNA o No host cells or vectors are needed o Concept: § Add many copies of primers that flank the gene of interest § Makes many copies of one gene fragment o Steps: § Denature DNA with heat to separate double strands § Lower temperature to allow primers to bind (anneal) § Raise temperature, which allows for complementary strand to be made (extension) o Pick gene of interest by making your own primer o Starting materials § Template DNA • Contains region that needs to be amplified § Oligonucleotide primers • Complementary to sequences at the ends of the DNA fragment to be amplified • Synthetic and are about 15-20 nucleotides long § Deoxynucleoside triphosphates (dNTPs) • Provide the precursors for DNA synthesis § Taq polymerase • DNA polymerase • Thermostable enzyme is necessary because PCR involves heating steps that inactivate most other DNA polymerases o Copying n § 1 à 2 à 4 à 8 à 16 à 2 o PCR is carried out in a thermocycler, which automates the timing of each cycle § all of the ingredients are placed in one tube § the experimenter sets the machine to operate within a defined temperature range and number of cycles o the sequential process of denaturing-annealing-synthesis is then repeated for many cycles § a typical PCR run is likely to involve 20 to 30 cycles of replication • takes a few hours to complete 20 § after 20 cycles, a target DNA sequence will increase 2 fold § after 30 cycles, a target DNA sequence will increase 2 fold o Can’t always use PCR because it doesn’t express gene or make protein o Can amplify one sequence out of a complex mixture o Researcher must have prior knowledge about the sequence of the template DNA § This is required to construct the synthetic primers o Can amplify nonspecifically by using random sequences § Mixture of primers with many different random sequences is used § These will anneal randomly throughout the genome and amplify most of the chromosomal DNA § Used to amplify very small samples, such as crime scenes o PCR is used to detect and quantitate the amount of RNA in living cells § Reverse transcriptase PCR or “RT-PCR”: • Isolate RNA from a sample • Mix RNA with reverse transcriptase and primer that will anneal to the 3’ end of the RNA of interest • This generates a single-stranded cDNA which can be used as a template DNA in conventional PCR • Figure 20.7 • Extremely sensitive: very little RNA needed to be detected • Needs two different primers o Primer needs to be added to the left of poly-A tail (where you want to amplify) o One primer at 3’ end and one at 5’ end § Real Time PCR: used to quantitate the amount of a specific gene or mRNA • Carried out in a thermocycler that can measure changes in fluorescence emitted by detector molecules in the PCR reaction mix • Can be used to quantify amount of DNA/RNA present • Taqman system uses a detector oligonucleotide that has a fluorescent reporter molecule at one end and a quencher molecule at the other end o Due to the proximity, the quencher molecule blocks the fluorescence of the reporter molecule on the oligonucleotide o During primer extension, Taq polymerase 5’-3’ exonuclease activity digests the detector oligonucleotide, separating reporter and quencher o Fluorescence will increase in proportion to the amount of PCR product produced § Steps: • Forward primer binds • Taq polymerase begins synthesizing strand • Taqman probe binds complementary to template DNA • Taq polymerase reaches probe and chews off the reporter, which is released and can no longer be quenched o Quencher no longer can absorb fluorescence o Fluorescence is observed § With high amount of DNA, fluorescence threshold is reached quickly because the reporter has been chewed off by Taq polymerase • This can be used to determine how much DNA/RNA was in original sample § Cycle threshold depends on amount of DNA initially • If there is a low amount of DNA/RNA, threshold reached slowly • If there is a high amount of DNA/RNA, the fluorescence threshold is reached quickly § When reagents are not limiting, product doubles with every cycle § Reaction fails as reagents become limiting and plateaus when one or more are used up § Cycle threshold is an amount of fluorescence in which you can detect it against the background § Compare unknown sample with a known standard • That is how you figure out how much DNA or RNA is in your sample - DNA sequencing o Underlies all aspects of inherited traits § Enables researchers to determine the base sequence of DNA o Dideoxy sequencing § DNA polymerase connects adjacent deoxynucleotides by covalently linking the 5’-P of one to the 3’-OH of another § Dideoxyribonucleotides (ddNTPs) are missing this 3’ OH group § Can be added to DNA, but nucleotide cannot be added to it after it has been added • Leads to chain termination § Many copies of primer and DNA are needed § Every once in a while a ddNTP is added to the complementary strand so strand stops growing • Only a low concentration of ddNTPs are added so chain termination only happens occasionally § Get a mixture of many DNA fragments that have been terminated with a ddNTP • Each ddNTP has a fluorescent color § Can “run” the fluorescent ended strands on a gel to separate them by length • Determine length from this • Shorter DNA runs faster • Each ddNTP has its own color • Fluorescent detector detects color and reads the peaks as the bands run off the bottom of the gel § Procedure is automated using a laser and fluorescence detector • The fragments are separated by gel electrophoresis o The mixture of DNA fragments are electrophoresed off the end of the gel o As the band comes off the bottom of the gel, the fluorescent dye is excited by the laser o The fluorescence emission is recorded by the fluorescence detector o The detector reads the level of fluorescence at four wavelengths Chapter 21: - Biotechnology: Technologies that involve using living organisms or their products to benefit humans. o Not a new innovation o In the 1970s molecular genetics helped provide new ways for organisms to benefit humans o GMO is an example o Organism that integrates recombinant DNA into its genome is transgenic o Always has safety concerns and often a negative public perception - Insulin: Regulates many physiological processes o Such as glucose uptake into fat and muscle cells o Made by Beta cells in the pancreas o Insulin-dependent diabetes is a B cell defect § Cannot synthesize enough insulin § Can use cows and human cadavers for insulin § Or recombinant bacteria • Make A chain recombinant (with fusion protein and B galactosidase so to not be degraded) • Also make B chain • Cleave off B galactosidase, purify A and B • Bond A and B together o Insulin is a hormone composed of two polypeptide chains, called the A and B chains - Genetically modified animals o Production of transgenic animals is a new area of biotechnology o Gene addition vs gene replacement § For stable inheritance, DNA must be incorporated into genome, which occurs by recombiantion § Gene replacement: Cloned replaces the normal • If cloned gene prevents gene from working it is a knockout § Gene Addition: Add cloned gene AND keep normal gene o In bacteria and yeast, gene replacement is common outcome § These have relatively small genomes, so homologous recombination occurs at a relatively high rate o In complex eukaryotes gene addition is extremely likely § These have very large genomes, so homologous recombination is rare o Have made mice with gene replacements § Must first select cells that carry out gene replacement (not addition) • Cells in which homologous recombination has occurred are preferentially selected § Cloned gene must contain 2 selectable markers • Nepmycin-resistant gene (NeoR) is inserted into the center of the coding sequence of the target gene • Thymidine-kinase gene (TK) is inserted adjacent to target gene o NOT WITHIN o Renders cells sensitive to killing by a drug called gancyclovir • The modified target gene is then introduced into mouse embryonic cells which can be grown in the lab § Figure 21.5 § Use stem cells in media with gancyclovir • genetic addition cells will have TK present and are killed on plate • Gene replacement cells will survive both the neomycin and the ganciclovir o Because they do not have the TK gene § These surviving (gene replacement) cells are inserted into blastocysts (embryonic cells) • Blastocyst has both modified cells and normal cells • à Chimera • modified and normal cells can mix together to create a single organism o mouse will have patches of dark fur and white fur (chimera) o Gene knockouts reveal the function of a gene and help scientists understand human diseases § Sometimes there is no obvious phenotype • gene has small contribution to overall phenotype • Another similar gene may compensate for gene function (gene redundancy) § Knockouts can teach us about human diseases • Mice and humans share many genes • Seeing characteristics of knockout mice may lead to treatments of human diseases • Have been useful in understanding cancer, obesity, heart disease, diabetes and many inherited diseases o Can do a gene knockin § Gene addition in which a gene of interest has been added to a particular site (noncritical) in the genome § Must make sure adding gene does not disrupt any other genes § Added to noncritical site/region § If there is a phenotypic effect of a gene knockin, it is usually representative that the gene is within the genome - Transgenic Livestock: § Not only cows o Can include the production of medicines in the milk of these animals § Sometimes called medical pharming o Look at Figure 21.7 o Often has very high yields o Helps when bacteria can’t do the full function of making the product (such as lacking protein folding ability) - Reproductive Cloning o Reproductive cloning refers to producing two + genetically identical individuals § Easier in plants b/c it can be done in somatic cells o Identical twins are genetic clones from one fertilized egg o Procedure § Start with a somatic cell § Remove nucleus from another unfertilized egg § Add somatic cell into the egg § Must make egg think it has been fertilized • Stimulate development § Cell then divides § Insert into surrogate § Pregnancy occurs - Stem Cells o Supply the cells that construct our bodies from a fertilized egg § Also replenish damaged cells in adults o Two characteristics § Capacity to divide § Can differentiate into one or more specialized cell types • Totipotent: Can become all cell types o Fertilized egg • Pluripotent: can differentiate into almost all types of cells but can’t give rise to an entire individual o Blastocyst o Fetus germ cells • Multipotent: can differentiate into several cell types o Adult stem cells • Unipotent: can differentiate into only one cell type o Adult stem cells (sperm in males_ o When a stem cell divides, one may remain undifferentiated, while the other can differentiate into a specialized cell type § The population of stem cells remains constant o Adult stem cells are typically multipotent or unipotent o In mammals, stem cells are commonly categorized based on their developmental stage and their ability to differentiate o Importance § Help understand genetic mechanisms that underlie the process of development § Offer the potential to treat human diseases or injuries that cause cell/tissue damage
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