13 Chap 9 and 8-1

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Unformatted text preview: Office hrs: Tues & Thursday 4:00-5:00 pm. jkwak@umd.edu Techniques in Cell and Molecular Biology! ! Chapter 9: Visualizing Cells! Chapter 8: Manipulating Proteins, DNA, and RNA! ! ! Chap 9: light microscope, radiosotope Chap 8: 1.cell culture, hybridomas, and ab 2. purifying and analyzing proteins 3. analyzing and manpulating DNA 4. studying gene expression and function. The is a chain of consequences.. Facts & Current Problems! ! ! Hypothesis and Idea! ! ! Experiments! ! ! Conclusion and New facts! Professor showed Fly Embryo Development SEM image the embryo gradually develops into the a fly. Light Microscope! ! Any light microscope has these lenses: (1) Condenser lens: illuminates the specimen.! (2) Objective lens: collects light rays focused on the specimen.! (3) Ocular lens: forms an enlarged and virtual image.! ! These lenses complement each other. Total magnification: the product of the magnifications produced by objective lens ! and the ocular lens! 1 ! Resolution! !  Changing oculars (5X to 10 X) increases magnification & resolution.! !- Switching to a 20X ocular lens? ! ----- does not provide additional detail: empty magnification! Resolving power: the ability to see two neighboring points as two ! ! ! distinct entities.! Resolving power is limited by 1. diffraction and wavelength of light. !- is limited by diffraction. ! !- The resolving power of a microscope is ultimately limited by the ! wavelength of light.! If two things have the same refraction index then it will disappear in the solution of the other. Visibility = Contrast! 3 problems: : difference in appearance between an object and its background! ! (i) a glass bead that is dropped into a beaker of immersion oil having ! !the same refractive index as glass will disappear from view!! ! (ii) Transparent objects are difficult to see.! !: Can overcome by staining specimen (increasing visibility).! ! (iii) Stains can t be used with living cells (toxic!).! People develop phase contrast microscopy and fluorescence microscopy to solve these problems. Phase-Contrast Microscopy! ! 1. Phase-contrast microscope! i)  Makes transparent objects visible ! !: by converting differences in refractive index ! ! into differences in intensity! iii) Useful for examining intracellular components of living cells.! ! iv) Has optical handicaps that result in loss of resolution.! ! v) Images suffer from interfering halos and shading.! 2 ! 2. Differential Interference Contrast (DIC)! !(Nomarski) microscope! ! i)  Minimizes the optical artifacts by achieving a separation of direct and diffracted beams.! ii) Produces images that has a three-dimensional quality.! B produced by phase contrast microscope can see the inside. Fig. 9-8 Light micrographs of ! (a) bright field, (b) phase-contrast, (c) DIC optics, (d) dark-field microscopy! Fluorescence (FL) Microscopy & related FL-based techniques! ! !  Fluorochromes (fluorophores)! : Absorb invisible UV radiation! In order to observe using !uorescence we need to have !uorochromes. FL microscopy is used for: i) Protein detection (Immunofluorecence)! !: fluorochromes conjugated to an Ab determine protein location ! ii) Location of DNA and RNA! iii) An indicator of membrane potentials! iv) Sensors for small molecules! ! A fluorochrome can absorb a wavelength of light. When fluorochrome absorbs light it gets exicted and the molecule becomes not stable, it wants to to becomes stablzed. So Fluorochrome release a portion of energy in the longer, visible wavelength = Fluorescence. Optical system of a fluorescence microscope Two filters and one splitting mirror in addition to the lens mentioned. This object or specimen contains fluorochrome. This filter So a longer wavelength is produced, yellow in allows only a range of this case. wavelength to pass through 450 - 490 nm. 3 ! One of the fluorochrome widely used is the green fluorescent protein originated from Jellyfish. Green Fluorescent Protein ! from Jellyfish! Three people discovered these: Osamu Shimomura, Martin Chalfie, and Roger Tsien share nobel price in 2008. Osamu Shimomura, Martin Chalfie, Roger Tsien! NP, 2008! !  GFP (Green Fluorescent Protein)! i)  Isolated from jellyfish! ii)  Protein localization and dynamic activities ! !---- GFP fused to the protein or the promoter under study! ! III) GFP fused to a promoter --Visualize where the gene is expressed. Use DNA cloning and recombination techniques we can incorporate GFP into the DNA, after the coding region, we want to light up. We can even use GFP protein to monitor dynamics in a living cell. We can replace coding region of a gene with GFP, the promotor contains when and where the gene is expressed, so when GFP shows where and when our gene is expressed. !  GFP (Green Fluorescent Protein)! i)  Isolated from jellyfish (Aequorea victoria).! ii)  Protein localization and dynamic activities ! !---- GFP fused to the protein or the promoter under study! ! iii)  GFP variants: YFP, CFP, RFP! E.g. Yellow fluorescent proteins….. (R. Tsien, UC San Diego)! Can use these to do many interest things. 4 ! !  GFP (Green Fluorescent Protein)! i)  Isolated from jellyfish (Aequorea victoria).! ii)  Protein localization and dynamic activities ! !---- GFP fused to the protein or the promoter under study! ! iii)  GFP variants: YFP, CFP, RFP! iv)  FRET (Fluorescence Resonance Energy Transfer):! !- use the GFP variants! !- to study protein-protein interactions! !- to measure changes in small molecule concentration ! !(sensors for calcium, cGMP, cAMP, amino acids, sugar, etc)! R. Tsien developed this. Use Fret to do these. FRET-based Yellow Cameleon ratiometrically reports changes ! in cytosolic calcium concentration! It has CFP and YFP 535/480 nm Ratio Yellow cameleon Calmondulin combines w/ Calcium ion. can be in two There is a space molecule b/w Calmodulin and M13. conformations depending on Exicted the concentration and emitts Fret allows YFP to emits of Questions: 535nm. Ca+. 1,200 total yellow cameleon We always measure molecules the emission of two Chemistry & distance! Ratio 535 nm/ 480 nm is 2, wavelengths. The They get close to each other. so 800 in conformation 2, and 400 in ratio can tell us conformation 1. something. 535 nm is coming from YFP, that means that there is higher Ca2+ concentration. 2 3.0 4 2.5 5 min 5 3 1 2.0 10 µM ABA 1 2 3 4 5 By measuring the wavelength and calculation the ratio of 535 nm to 480 nm we can deduce the Ca2+ concentration. [Ca2+]cyt Transgenic guard cells expressing Yellow Cameleon! To summary: Techniques use to visual live DIC, phase contrast, use GFP help visual where and when is expressed. Using Fret we can sense the concentration of small molecules. 5 ! Video microscopy & Image processing! ! ! Advantage for viewing specimens ! -  allow video cameras to image specimen at very low illuminations! (i) imaging live specimens labile to heat.! (ii) fluorescence specimens that are easily bleached.! ! Use video microscopy and image processing b/c the problems above. his is a problem: ! !Confocal scanning light microscopy! Another technique that we currently use. Light microscopy: background light rays interfere with the light rays from the plane of focus.! -  Marvin Minsky at MIT! Allows only light coming from the specimen but not the background. -  Fluorochromes (in the specimen)! -  Laser! -  dichroic mirror ! (FL with longer wavelength passes through)! -  pinhole aperture.! Allows light w/ longer wavelength to pass. (Fluorochrome)! Light Microscope! Light microscope image from specimen and background. By moving this up and down we can have different images. Confocal Microscope! -  Out-of-focus information reduced! -  Good for thick specimens! -  Optical section! -  3-D reconstruction by stacking 2-D optical sections collected in series! Image from background will be significantly reduced. Move focal plane, see different images. E.g. thick specimen. 6 ! Radioisotopes! Another thing to visualize things. ! ! Isotopes: Atoms having the same number of protons & ! different numbers of neutrons are said to be isotopes.! -  are radioactive when they contain unstable combination of protons and neutrons. !! -  When disintegrating, they release particles or electromagnetic radiation.! !! ! (i) " particle: equivalent to the helium nucleus (2 protons + 2 neutrons)! (ii) # particle: equivalent to an electron! !- liquid scintillation spectrometry: measures the amount of ! !radioactivity in the sample! !- autoradiography: to localize sites containing radioisotope! (iii) $ particle: consists of electromagnetic radiation or protons.! Three particles that isotopes release. 32P 35S 33P ! ! 25.3 d, ! Beta particle! The half-life is the time for half the radioactive nuclei to undergo radioactive decay. ! ----- After two half-lives, there will be one fourth the original sample.! Procedure involve isotopes. To visualize proteins incubate cell treat cell w/ radioactive particles. put cell on slide Radioactive sensitive. Put it in liquid emulsion store in box view. Chromosome! (microscope)! RNA labeled! with 3H-Uridine! (autoradiogram)! 7 ! Light Microscopy Lenses Resolution, visibility (contrast) Phase contrast microscope, DIC (nomarski) Microscope Fluorescence Microscope: fluorochrome, fluorescence, GFP, FRET (Ca2+/Yllow cameleon), cGMP sensor? depends on your creativity. Video microscopy Confocal microscopy Radiosotopes Detection methods for radioactivity are so sensitive. They are so sensitive, small number of molecules can be viewed. Cell separation technique: FACS (fluorescence activated cell sorting) Isolate the protein in your mind. Cells pass through a nozzle fluorescence = negatively charged. !  Droplets containing single cells are given a (+) or a (-) charge, depending on whether or not the cell is fluorescent. not fluoresence = positively charged. !  Cells are deflected by an electric field into collection tubes based on their charge. use this machine to select certain types of cells. Cell Culture! ! : Growing cells outside an organism! - cultured cells can be obtained in large quantity.! - contain only a single type of cell.! - cells can differentiate in culture.! - respond to treatment with drugs, hormones, and growth factors.! ! ! ! -  primary culture! !: cells that are obtained directly from the organism (usually embryo) ! !
 - Secondary culture! !: cells that are derived from a previous culture ! ! !! People have developed ways to maintain single type of cells. cells are outside of host, but they can still differentiate and develop. ! !  Cell line! !: cells that have undergone genetic modifications that allow ! ! them to grow indefinitely.! -  some are derived from tumors or cells treated with carcinogenic viruses or chemicals.! !  Embryonic Stem (ES) cells! (i) derived from an embryo! (ii) can be kept in cell culture as ! an immortal cell line! (iii) can differentiated into specific ! cell types (hormones, growth ! factors)! Cells in culture do not grow eternally, this is a problem. People so developed cell lines to make cells grow indefinitely. treat them with carcinogenic chemicals, or viruses, or stimuli so they can grow indefinitely. 8 ! -  Plant cell culture! !: Celullase to remove cell wall and produce protoplasts ! !: can grow in a defined medium.! !----> grow into a callus (undifferentiated clump of cells) ! !then to a plant (regeneration --- stem cells!)! ---> roots & shoots induced. most of plant cells can be differentiated into callus and be regenerated. ways to visualize proteins. Antibodies and Hybridomas! ! !  Antibodies: proteins produced by lymphoid tissues in response to the presence of foreign materials (antigens)! -  -  ! ! Remarkable specificities: e.g., antibodies can distinguish two peptides that differ by only a single amino acid!! !! polyclonal and monoclonal Abs! (Ser vs. phosphorylated Ser) Two types or antibody. Advantage: antibodies are so specific, so we can differentiate. They can even differentiate proteins that are phosphorylated or not phosphorylated. !  Preparation of Antibodies! (i)  Polyclonal antiserum! -  -  -  an animal is repeatedly injected with antigens.! Obtain whole blood and remove cells and clotting factors to produce antiserum.! further purify to get the immunoglobins ! ! -  These Abs are polyclonal.! !: bind to the same antigen but have different V regions! V regions are antigen recognition regions. 9 ! B-lymphocytes do not grow and devide in culture. (ii) monoclonal antibodies! !: produced by a single clone! ! -  Use (i) cells derived from a single B-lymphocyte producing Ab with the same Ag recognition region.! -  Myeoloma cells do not have HGPRT. C. Milstein & G. Köhler of MRC of the UK in 1975.! -  Use (ii) cancer cell lines because Ab-producing cells do not grow and divide in culture.! -  We only want hybridomas, this is the enzyme selecting only hybridomas. Combines the properties of normal lymphocyte and immortal !myeloma by fusing the two cells (hybridomas).! , resulting in hybridomas. ! -  HGPRT allows only hybridomas to survive in the HAT medium.! (hypoxanthineguanine phosphoribosyl transferase) So myeoloma cells cannot grow in HAT medium. Hybridomas will die before 3 three days b/c they have the enzyme. Myeloma cells are HGPRT-mutants spleen removed Hybridoma In polyethylene glycol This is how we generate mono antibody, which can be used to distinguish serum vs. phosphorylated serum. Selection of cells in HAT medium b/c they have HGPRT. * Test supernatant for presence of desired Ab Antibodies can do many things. -  Antibodies are widely used in ! (i)  Immunoprecipitation! (ii)  Western blot! (iii)  Immunolocalization! (iv)  Immunofluorescence! protein visualization. Immunoprecipitation: antibody form complex, that precipitate. Immunolocalization: tissue section, visualize proteins in the tissue. Immunofluorescence: use antibody to do fluorescence microscope. 10! Fractionation of a cell s contents by differential centrifugation! --- organelles have their own density in a centrifugal field! Do centrifugation to separate cell organelles. Use low speed gradually to high speed to separate massive organelles first, then separte lighter organelles. One type of centrifugations. Velocity Sedimentation Centrifugation! --- useful for fractionation of subcellular components! --- separation by size and shape! Gradient is set b4 the centrifugation. There is a gradient in the centrifugation tube. Equilibrium sedimentation Centrifugation! --- separation on the basis of buoyant density.! - -- useful for protein & nucleic acid separation! - -- The tendency for Cs to be concentrated toward the tube bottom! : counteracted by the tendency to become redistributed by diffusion.! DNA + CsCl in soln No gradient first, but it is generated after contrifugation. AT rich DNA molecules are less dense than GC rich DNA molecules, b/c GC rich molecules have one more After centrifugation H-bond. we can separate DNA molecules. 11! Fractionation of the tube contents.! Separation of plasmid DNA! Use syringe to separate. Purification of proteins's ! ! purposes: ----> to study the structure and function of the protein! Stucture can predict function. Understanding the structure we can predict the interaction. Purification of proteins! Purification is measured by specific enzymic activity. : to study the structure and function of proteins.! ! -  Protein purification is measured as an increase in ! !specific activity (enzyme activity/total amount of protein)! ! ! ! Methods to purify proteins: Selective precipitation Column chromatography Polyacrylamide Gel Electrophoresis 12! At one point, there is no more activity b/c precipitation stopped the protein activity. ! 1.  Selective Precipitation! -  -  based on solubility differences of proteins.! solubility of a protein depends on the distribution of hydrophobic and hydrophilic side chains on its surface.! -  -  yields a large increase in specific activity.! achieved by gradual addition of salt! !---- a loss of activity in the soluble fraction when the protein precipitates.! -  ammonium sulfate: highly soluble, high ionic strength! Percent Maximal Acitivity. (NH2)4SO2. After precipitation, we do this. 2. Liquid column chromatography! -  utilizes some type of porous matrix! 3 types of chromatography: (i) Ion exchange chromatography! ! (ii) Gel filtration chromatography! ! (iii) Affinity chromatography! (1)  Ion-Exchange Chromatography! -  based on ionic charge of protein which is dependent on the pH.! -  pI is a pH. Isoelectric point (pI)! !: pH for a protein at which the protein is neutral,! ! b/c the total number of (-) charges equals the total number of ! ! (+) charges.! Any proteins can be charged based on the pH of the soln. ! ! Two most common resins are! ! (i) diethylamionethyl (DEAE)-cellulose (Cellulose-O-CH2CH2-N+(C2H5)2 )! : anion exchanger ! --- DEAE is positively charged and thus binds negatively charged molecules.! ! (ii) carboxymethyl (CM)-cellulose (Cellulose-O-CH2-COO-)! : cation exchanger! --- CM is negatively charged and thus binds positively charged molecules.! 13! Elution buffer! Protein elution carried out by ! (i) increasing the ionic strength! and/or 
 (ii) changing pH.! Separte proteins based on their sizes. (2) Gel Filtration Chromatography (size exclusion chromatography)! -  separates proteins (or NA) on the basis of their effective size.! -  resin: polysaccharides (dextrans or agarose) of different !! ! porosity (molecular sieve) ! -  Porosity allows proteins to diffuse in and out of the beads.! Resin acts as sieve. porous. proteins go in and go out. Larger proteins will not go in and will flow down. Method to determine the mass of a protein. (3) Affinity Chromatography! ! -  based on the structural properties of a protein (physical interaction) ! : enzymes & substrates! based on function and interaction. receptors & ligands! Ag & Ab ! DNA & DNA binding proteins (RNAP, Txn factors)! Elution buffer! 14! (3) Affinity Chromatography! ! √ Elution by ! (i) changing ionic strength! (ii) pH changes ! (iii) adding excess free ligands.! ! √ Can achieve a near-total purification ! in a single step!! ! √ Used to purify recombinant proteins! -  a specific purification tag is fused to the! protein of interest to create ! a fusion protein! Determining protein-protein Interactions! ! ! Techniques for determining protein-protein interactions! (i)  FRET! (ii)  Affinity chromatography! (iii)  Immunoprecipitation (Ab-protein A-protein B)! (iv)  Yeast two hybrid! -  Stanley Fields &Ok-Kyu Song at SUNY Stony Brook, 1989! -  Depends on ! !(a) protein-protein interactions! !(b) reconstitution of a functional transcription factor! !(c) expression of a reporter gene such as #galactosidase (lacZ) mediating color change reaction.! -  A transcription factor is divided into two domains: a DNA-binding domain & an activation domain.! -  lacZ expression depends on a functional transcription factor.! ! -  The DNA binding domain linked to the bait protein under study ! ---> look for interacting proteins!! Yeast cell turns blue! 15! In White colony! In Blue colony! Detection of protein-protein interaction based on FRET! 3. Polyacrylamide Gel Electrophoresis (PAGE)! Electrophoresis! : depends on the ability of charged molecules to migrate ! in an electric field. ! 16! !  PAGE! -  -  Molecular Sieve: acrylamide & bis-acrylamide are cross-linked! Proteins negatively charged b/c PAGE is carried out using alkaline buffers (note: not uniformly charged!)! -  Migration depends on ! (i)  Charge density (charge per unit of mass)! (ii)  Shape! (iii)  Size! -  -  Coomassie blue or silver stain.! Gels can be autoradiographed or ! !transferred to membrane for immunological detection (Western blot)! SDS-PAGE! : PAGE in the presence of SDS (sodium dodecyl sulfate)! -  SDS binds to a protein (1.4g/g protein) and makes proteins unfold into a rod-like shape! -  Proteins have equivalent charge density! -  Migration depends only on the mass (size)! -  Can determine the molecular mass! ! (2) Two-dimensional Electrophoresis! ! -  Patrick O Farrell (UCSF), 1975! -  pI- and molecular mass-dependent! -  Separation by isoelectric focusing (pI, 1-D), ! and then by SDS-PAGE (size, 2-D).! 17! IPG Strip! Protein samples in buffer! +! 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5! -! 1-D separation (pI) ! by IEF ! Proteins separated on IPG strip! Place the strip onto a well:! 2-D separation (size) by SDS-PAGE ! +! -! -  Proteins differentially expressed! -  Changes in the proteins present in a cell under different conditions! -  Changes in the proteins present at different developmental stages ! -  Changes in the proteins present at different cell cycles! condition A! condition B! 4. Mass Spectrometry! - Determines ! (i)  Molecular mass! (ii)  chemical formulas ! (iii)  molecular structures! ! - Converts into positively charged, gaseous ions.! - A magnetic field separates charged ions according to molecular mass.! - Smaller ions travel faster and strike the detector more rapidly. ! 18! ! In order to identify unknown proteins ! (i) Proteins are digested by trypsin (C-terminal side of Arg & Lys)! (ii) The peptide is identified by a database search (pattern match)! ---> Lysozyme!! Purification, Analysis, and Manipulation of nucleic acids! Extraction of NA from cells! ! (i)  Detergent (SDS)! -  Lyses the cells and nuclei and thus releases DNA! -  Inhibits nuclease activity.! (ii)  Phenol removes proteins from the sample.! (iii) !Cold Ethanol precipitates NA.! Separation of DNAs by gel electrophoresis! (i)  PAGE: smaller DNAs (< a few hundred bp)! (ii)  Agarose GE: Large DNAs ! (iii)  Pulsed-field GE: Larger DNAs (> 25 kb) (direction of the electric field in the gel is periodically changed).! EtBr-stained! 19! Measurements of protein and nucleic acid concentration! ! ! ! Protein or NA conc. ---- Spectrophotometer measures the amount ! ! ! ! ! ! ! ! ! of light of a specific wavelength that is absorbed.! !- Proteins show maximal absorbance at 280 nm ! ! ! ! !--- because of Tyr, Trp, and Phe.! !- NAs have maximal absorbance at 260 nm.! NA hybridization! ! -  ssNA molecules of complementary base sequence can form a ds hybrid!! ! ! ! Separation of DNA-RNA hybrids from ssDNA! -  Hydroxylapatite column! !: ds hybrids binds to the Ca2+ phosphate salts in the column ! while ssDNA molecules pass through.! 1.  Detection of NA! --- Southern blot (DNA transfer; named after Edwin Southern)! --- Northern blot (RNA transfer)! 20! !  Radioactive DNA probe preparation! (i)  Nick translation: DNase I and DNA polymerase I introduce and move ss nicks in ds DNA by replacing nucleotides with radioactive ones.! (ii) Random priming: uses random hexamers as primers for DNA duplication (radioactive nucleotides)! ! (iii) End labeling (5 ends, Polynucleotide kinase)! ! Random Priming End Labeling Other NA hybridizations! ! - in situ hybridization ! ! - Microarrays (cDNA- or oligomer-based chips).! Gene expression ! in Arabidopsis embryo! Myogenin ! in mouse embryo! FISH ! (FL in situ hybridization)! 21! Recombinant DNA technology! ! ! Recombinant DNA! : molecules containing DNA sequences derived from more than one source ! (chimeric DNA).! 1. Restriction Endonucleases (REs)! - cleave dsDNA! - found in bacteria: function to destroy viral DNA! -  Recognize 4 to 6 nucleotide sequences! -  Recognition sequences are palindromic ! (e.g. Eco RI).! -  Blunt ends or Protruding ends. ! - host cells protect their DNA from the attack by methylating the DNA!! 2. Formation of recombinant DNAs ! -  use vectors as a vehicle! -  Plasmids: small, circular, ds DNAs that are different from the -  chromosomes.! -  Cut & Paste (REs & DNA ligase)! ! ! The first rec DNA by P. Berg, H. Boyer, A. Chang, & Stanley Cohen at Stanford and UCSF in 1973.! 3. DNA cloning! -  A technique to produce large quantities of a specific DNA ! fragment (amplification!).! -  A pure form of any specific DNA fragment can be purified.! ! Vector DNA! - a vehicle for carrying foreign DNA into a suitable host cell. ! -  Contains necessary sequences (see next slide)! 22! Cloning eukaryotic DNAs in bacterial plasmids! 1. Plasmids! (i) origin of replication! (ii) marker gene(s) that make the cell resistant to antibiotics! (iii) multicloning sites! 2. Transformation! -  Rec DNAs: introduced to bacterial cells (competent cells) ! (i)  Heat shock! (ii)  Electric shock (electroporation)! (iii)  PEG! ! Cloning eukaryotic DNAs in phage genomes! ! bacterophage %! -  a cloning vector! -  the genome is a linear ds DNA (~ 50 kb)! -  The dispensable middle fragment can be replaced (~ 25 kb)!! -  Rec DNA is packaged in vitro (non recDNA is too short to be packaged).! !  Formation of a DNA library! DNA library: collections of cloned DNA fragments! ! (i)  genomic DNA library: contains all of the DNA sequences of the species! (ii)  cDNA library: DNA copies of an mRNA population.! ! -  Genomic clones provide:! (i) regulatory sequences (promoter etc)! (ii) genomic structure: noncoding sequences flanking the coding region! 23! Cloning larger DNA fragments in specialized cloning vectors! ! (1) YAC (Yeast Artificial Chromosome)! - can accept DNA fragments up to 1,000 kb! - Contains all of the elements of a chromosome (several replication origins, telomeres, centromere, selection markers, etc)! (2) BAC (Bacterial Artificial Chromosome)! - up to 500 kb.! - specialized plasmids (F factors) that contain a bacterial replication origin ! - advantages over YAC:! (i)  Short generation time! (ii)  Higher transformation efficiency! (iii)  Easier DNA preparation! ! cDNA libraries! -  contain millions of different cDNA clones ! ----> to be certain that the rarer mRNAs will be represented.! ! -  Required enzymes! !(i) Reverse transcriptase ! !(ii) RNase H! !(iii) DNA polymerase I! ! 24! Amplification of DNA by PCR! PCR (Polymerase Chain Reaction)! ! -  Kary Mullis of Cetus Corp. in 1983.! -  Taq polymerase (heat stable DNA polymerase) ! -  DNA template! -  dNTPs! -  primers! -  Thermal cycler (PCR machine)! ! ! (NP, 1993 for the discovery)! ! !Typical PCR conditions! ! 94ºC, 4 min ! ! ! !------ 1 cycle! ! 94ºC, 30 sec (denaturation)! 55ºC, 30 sec (annealing) ! !------ 35 cycles! 72ºC, 2 min (polymerization)! ! 72ºC, 10 min (extension) ! !------- 1 cycle! ! !In Thermal Cycler (PCR machine)! 25! PCR cycle no Protein kinase Alcohol oxidase UBQ WT Mutant 1 Mutant 2 DNA sequencing! ! Restriction enzymes, DNA cloning, and a new sequencing methodology facilitated DNA sequencing and current molecular biology.! !(i) Maxam and Gilber chemical method! !(ii) Sanger s polymerization termination method (enzymatic)! !(iii) Next-gen sequencing! ! ! ! ! 1. Allan Maxam and Walter Gilbert (Harvard U) ! : a chemical approach to determine DNA sequence.! ! 26! 2. The Sanger-Coulson (Chain termination) method! ! -  DNA template! primer! DNA pol I! dNTPs (one of them is radiolabeled)! ddNTPs! - The incorporation of ddNTPs is infrequent and random, resulting in immature DNA polymerization.! ! ! -  Resolve the sequencing reaction products in a PAGE gel.! ! ! -  Fredrick Sanger sequenced a virus genome (~ 5.4 kbp) in 1977!! In A (ddATP) reaction! DNA template (millions of molecules)! DNA polymerization! ! Termination! ddA! ddA! ddA! ddA! ddA! ddA! 27! Fluorescent-labeled ddNTPs with different colors allow one to fractionate the sequencing products in one lane.! Studying Gene Expression and Function Gene transfer into Eukaryotic cells and mammalian embryos! : to study the expression of foreign genes and proteins! ! ! Gene transfer methods:! (i)  Transduction: viral-mediated gene transfer (retrovirus)! (ii)  Transfection: Transfer of naked DNA into cultured cells! - !chemical transfection: calcium phosphate or DEAE-dextran! - !electroporation: a brief electric pulse! -  lipofection: fuse with the lipid bilayer and delivers the DNA! (iii) !DNA microinjection! - !into the nucleus! -  Xenopus oocyte, ! -  mouse embryo, C. elegans! Determination gene function by gene elimination! (deletion of a gene can lead to the absence of a cellular process!)! ! (i) Forward genetics: learning about genotype by studying mutants phenotypes (from phenotype to gene).! (ii) Reverse genetics: determining phenotype (or function) based on the knowledge of genotype (from gene to phenotype).! Reverse genetics techniques! (i)  Knockout mutants (mice, plants, yeast, fly … etc)! (ii)  RNAi (RNA interference)! -  target mRNA is degraded due to the presence of a small ds RNA.! -  can examine the phenotype of the organism that results from RNAi (Knockdown)! 28! Transgenic animals and plants! ! ! Transgenic mice! -  R. Brinster (U Penn) & R. Palmiter (U Washington), 1981 ! : Transgenic mice with a rat growth hormone gene! Knockout mice! ! -  determines a gene s function ! -  ES (embryonic stem) cells 
 ! -  Homologous recombination b/n endogenous and the mutant genes.! -  M. Capecchi (U Utah),! O. Smithies (U Wisconsin), ! and M. Evans (Cambridge U) in 1980 s.! ! Generation of transgenic plants! : for Functional study & Genetic manipulation! ! (i) T-DNA transformation! -  Genes are cloned into the Ti plasmid of Agrobacterium! -  A section of Ti plasmid (T-DNA) is passed from the Agrobacterium to the plant cell.! ! (ii) Gene gun! 29! Transgenic mutant plants! (Dr. E. Meyerowitz, CalTech)! 30! ...
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