Molecular Biology MCDB 427
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Date Created: 10/29/15
You are responsible to prepare the material included here plus western blotting I ve also included some details for which you will not be responsible but may be curious about shown in italics I Molecular Separations Gel Electrophoresis A Principles of Gel Electrophoresis 1 DNA RNA and proteins all carry an electrical charge therefore they can move in an electrical eld 2 DNA amp RNA carry a net negative charge phosphates 3 Net charges on proteins differ 4 General procedure 5 The basis of a gel s ability to separate nucleic acids amp proteins ofdifferent sizes is based on friction B Types of gel material 1 Agarose a A highly purified form of agar a complex polysaccharide extracted from the marine red algae Gelidium or Gracilaria b When heated to boiling the polysaccharide molecules dissolve in an aqueous buffered solution amp form fully extended strands c When cooled the polysaccharide strands fold amp twist up with each other forming a meshwork d Primarily used in the analysis of DNA amp RNA e Resolution i Can effectively resolve DNA fragments ranging from 500 bp 20000 bps ii Concentration of agarose affects resolution 06 resolves 1 20 kb DNA 09 resolves 05 7 kb DNA 15 resolves 02 4 kb DNA 20 resolves 01 3 kb DNA 2 Polymerized acrylamide AKA polyacrylamide a Can resolve DNA fragments from 5 1000 bp used for DNA sequencing b Gel matrix of choice for almost all protein electrophoresis procedures 0 Polyacrylamide gels are formed by the copolymerization of acrylamide CH2CHCONH2 with a crosslinking agent to form a threedimensional lattice d Crosslinking agentis NN methylene bisacrylamide BIS CH2CH CONH CH2NHCOCHCH2 e polymerization reaction occurs by a free radical chain mechanism i uses ammonium persulfate amp tetramethylethylenediamine TEMED CH32NCH2 CH2NCH32 ii ammonium persulfate is the disulfate ester of hydrogen peroxide O3S OO 803 amp it readily changes into the unstable SO4 radical iii TEMED is a tertiary amine that reacts with these radicals to form TEMED free radicals which in turn react with acrylamide to induce polymerization f The average size of the pores in a polyacrylamide gel can be controlled by varying the percentage of acrylamide andor by varying the degree of crosslinking BIS Generally the BIS concentration is kept constant at 5 amp the percentage of acrylamide is varied to make gels of different porosity C Electrophoresis Buffers Electrophoresis is performed in buffers with de ned pH amp ionic strength D DNA electrophoresis Figure 51 1 Loading dye buffer a 50 sucrose to make the DNA solution very dense stays in the well prior to electrophoresis b quottracking dyesquot i Bromophenol blue migrates at approximately the same rate as a 05 kb fragment ii xylene cyanol migrates at approximately the same rate as a 5kb fragment 2 Wells are closest to the cathode 3 Negatively charged DNA migrates toward the anode 4 The DNA fragments are separated based on size 5 The gel is stained with the uorescent dye ethidium bromide 10 pgml destained with distilled water amp examined under ultraviolet light 6 A photograph film or digital is taken ofthe stained gel 7 Ethidium bromide is a at ringshaped molecule which binds to DNA by inserting itself intercalates between base pairs ofthe DNA double helix EtBr is a mutagenic compound and a suspected carcinogen 8 Methylene blue an alternate DNA stain a Nonhazardous amp can visualize the DNA fragments on a simple light box b Useful in teaching labs c Less sensitive amp requires longer staining amp destaining times 9 Known size standards quotladdersquot a The mobilities of the known size standards are plotted versus the log of their number of base pairs b Figure 52 E Pulsedfield gel electrophoresis PFGE 1 Analyzed on 1 agarose gel but with a modi ed apparatus a The pulsed eld apparatus has four sets of electrodes b The two sets of electrodes are set at an angle of 120 with respect to one another 2 During electrophoresis the DNA molecules are subjected to alternating bursts of current from the two electrode pairs pulling the DNA alternately to the right amp to the left as it advances through the gel 3 The DNA reorients itself each time the current is switched a The time it takes for reorientation is dependent on its length b Large fragments take longer 4 Figure 53 F Electrophoresis of Proteins 1 Proteins vary not only in size but also in shape amp charge 2 Treat all proteins with the detergent sodium dodecyl sulfate or SDS to quotdenaturequot the subunits a Two advantages over heat denaturing i Coats all the polypeptides with negative charges so they all have net negative charge amp will electrophorese toward the anode ii All polypeptides electrophorese according to their molecular weight b Fig 54 3 Dithioerythritol DTT is also used to reduce amp break disul de bonds 4 SDSPAGE a common method to separate proteins 5 Coomassie Brilliant Blue a common protein stain a Gel placed in a staining tray b Polypeptide bands are quot xedquot amp most of the SDS is removed by soaking the gel in a dilute acetic acid amp methanol solution c The gel is then soaked in a solution of Coomassie Blue dissolved dilute acetic acid amp methanol d Destaining 6 Silver staining a 10 to 100fold more sensitive but also more laborious amp expensive b Based on the chemistry used in photographic development i The gel is soaked in a dilute solution of methanol to x the polypeptide bands to the gel ii The gel is then incubated with an acidic solution of silver nitrate iii An image is formed by reduction of ionic silver to its metallic form by formaldehyde at alkaline pH sodium carbonate iv Development is stopped by acidifying the solution with acetic acid G Twodimensional gel electrophoresis 1 First step lsoelectric focusing a Old methoda protein mixture is electrophoresed through a narrow tube gel containing ampholytes that set up a pH gradient from one end ofthe tube to the other b New methodlmmobilized pH gradient IPG strips are used Each sample protein applied to an IPG strip and will migrate to its isoelectric point pl the point at which its net charge is zero 2 Second stepSDSPAGE a The gel is removed from the tube amp placed at the top of an ordinary SDSPAGE b Further resolved based on their molecular weights 3 Diagram oftwodimensional electrophoresis of proteins Figure 55 Southern Blots Identifying Specific DNA Fragments 1 1975 Dr Edward Southern 2 Figure 512 a DNA in the gel is denatured amp a nitrocellulose membrane is placed on top of the gel b Buffer 880 standard saline citrate NaClsodium citrate is wicked through the gel carrying the DNA fragments by capillary action amp transferring them to the nitrocellulose membrane 0 Baking the nitrocellulose the singlestranded nucleic acids are irreversibly bound to the membrane The membrane is then allowed to interact with the labeled singlestranded DNA probe that contains sequences for the DNA of interest d After incubation excess probe is washed away and the membrane is placed over Xray lm The radioactive probe will expose the xray lm revealing the position of restriction fragments that contain sequences able to hybridize with the probe sequences e Note that there are now many modi cations including the use of different membranes nylon and denaturation conditions 3 Stringency a Can selectively identify DNA sequences that are either a perfect match with the probe sequences or those that are related but not perfectly complementary to Fig 526 the probe sequences b Modifying stringency is done during the washing step by adjusting the temperature andor adjusting the ionic strength ofthe wash buffer Ill DNA Sequencing determining the exact nucleotide base sequences of DNA Based on the high resolution separation of polynucleotide fragments on polyacrylamide gels A Two methods both developed in 1975 1 MaxamGilbert procedure fragments are generated using chemical reagents which are speci c for each nucleotide 2 Sanger dideoxy procedure chaintermination sequencing a Polynucleotide fragments are produced by use of the enzyme DNA polymerase b Basis of automated sequencing B Sanger dideoxy chain termination procedure 1 Requires a singlestrand DNA amp a short synthetic deoxyribonucleotide primer complementary to a small portion ofthe single strand a Oligonucleotide primer 20 bases long b Primer is designed to hybridize to a sequence adjacent to the MOS region of the specific vector 0 Primer is designed such that it s 339 end will be pointing toward the insert DNA 2 The single strand serves as the template for DNA synthesis a New strands are extended from the 339hydroxyl group on the primer using deoxyribonucleotide triphosphates amp the Klenow fragment of DNA polymerase b dideoxyribonucleotide a nucleotide derivative lacking hydroxyl groups at both the 239 and 339 carbons added to the above reaction When a dideoxyribonucleotide is added the resulting chain can not be extended No 339terminal hydroxyl group to attach the next nucleotide 3 Generating quotnested setsquot gure 518 a Four DNA synthesis reactions are run in separate tubes