Final focuses heavily on lecture 20 25 but below is a quick summary of these lectures Besides this I suggest studying your old exams BSCI330 FINAL EXAM REVIEW Lecture 20 Manipulating DNA RNA and Proteins Cells retain morphological and functional characteristics in culture removed from body Embryonic stem cells can either o Reproductive placed in foster mother calf or o Therapeutic transferred from early embryo to culture dish o To differentiate ES cells gene regulatory proteins and other factors added Centrifugation o Low speed Pellet contains whole cells nuclei cytoskeletons o Particles higher density and larger in size travel at faster rate Hence faster the centrifugation speed the smaller particles settle Difference between different types of gels o Pore size ie Small acrylamide gel PCR This is a very important topic and likely question on Exam o Amplify DNA o Several cycles of changing temp to cleave and anneal o Each cycle 2X double stranded DNA produced Hypervariable microsatellites o Used DNA fingerprinting o Created by variable number of tandem repeats VNTR Ex 2 nucleotides A and C repeated variable number of times Generally di tri tetra nucleotides In noncoding DNA regions On each side of repeat are flanking regions of unordered DNA allows specific loci for primers to amplify the microsatellites with PCR Primers for PCR from these unique flanking regions Useful genetic markers o Highly polymorphic o Created through slippage mechanism Dideoxy sequencing aka Sanger method o 1 of many methods to sequence DNA o uses 2 3 dideoxynuclotide triphosphates ddNTPs this method takes advantage of the H bonded at the 3 hydrogen rather than usual OH bonded there o this sequencing terminates DNA chain elongation because can not form phosphodiester bond with next nucleotide o once completed PAGE gel electrophoresis performed sequence produced from this read from bottom to top to get sequence a can also do this with fluorescent dye detection b a b Restriction nucleases o Cut DNA o Blunt cohesive o Cohesive cohesive Ligation by DNA ligase o Recombinant DNA Cohesive ends to be ligated have to be capable of base pairing with each other Blunt ends can be ligated to other blunt ends Compatible ends do not have to be on different molecules Recombinant plasmid into bacterial cell cell culture millions of new bacteria purified amplified plasmid obtained from isolated lysed bacterial cells Human Genomic DNA library o Total genomic DNA from single organism o Recombinant human DNA plasmids in bacterial cells mRNA complement cDNA for analysis and storage o used to ID mRNA expressed in a tissue o converted to DNA because greater stability and DNA can be amplified by PCR or bacteria DNA molecules labeled with radioactivity or chemical modification invitro o end label radioactivity DNA labeled at 5 end with polynucleotide kinase and p labeled ATP Transferred phosphate is gamma phosphate o body label radioactivity denature and anneal with hexanucleotides add DNA polymerase and labeled nucleotides DNA polymerase incorporates labeled nucleotides resulting in a population of DNA molecules with labeled Ex Of a sequence on both strands Incorporated phosphate alpha phosphate o Chemical modification Antibodies and fluorescent markers Temperature changes Labeled probes RNA and DNA localization Southern Blot DNA Northern Blot RNA o First agarose gel electrophoresis o Then completed gel separated nucleic acids blotted onto nitrocellulose paper by suction of buffer through gel and paper o Nitrocellulose paper removed with tightly bound NAs o Radiolabeled probe hybridized to DNA in sealed plastic bag o Lastly labeled probe hybridized to complement DNA bands visualized by autoradiography SDS polyacrylamide gel SDS PAGE o Separates proteins on size o First proteins are heated with SDS and mercaptoethanol this surrounds proteins with negatively charge SDS molecules o Then separated in gel Isoelectric focusing IEF o Separates proteins by charge o Low pH positively charged o High pH negatively charged o Neutral pH isoelectric point where there is no change no migration in electric field 2D PAGE o combines IEF and SDS PAGE Protein separation o Colum chromatography Propterty separation through mesh o Ion exchange chromatography Charge separation o Gel filtration Size o Affinity chromatography Separates proteins using their specific interaction with other molecules o Combination of chromatography Highly pure preparation Detecting protein interactions o Yeast two hybrid system Recombines genes encoding bait and prey Introduced into yeast cell o Fluorescent Resonance Energy Transfer FRET Fluorescent res Energy transfer green light Fl Light detected protein activation X ray diffraction o Protein structure determination Nuclear Magnetic Resonance NMR o Protein structure determination and diagnostics of protein IN SOLUTION o BLAST Find similar proteins in solution clues to function of protein Basic local alignment search tool Genetic Mutations o Reveal identity of proteins control behavior Worm example with social and isolated eating Temperature sensitive mutations to study essential processes in cell o Isolated to study mutations in essential genes Genetics help detect function of genes o Working backward from the phenotype the organisms genotype is determined Lecture 21 RNA world RNA Hypothesis origin of life on earth o RNA today still regulate many processes o RNA can form secondary and tertiary structures Ribozymes o Catalyse chemical reactions Ie Specific RNA cleavage o We can make and select these through addition of ATP and derivative replacing oxygen like sulfur o Catalysis and replication RNA that can catalyze its own synthesis o Ribozymes subject to allosteric regulation By cooperative binding Theophylline and Flavin Mono Nucleotide FMN RNA world hypothesis origin of life according to Both required for self cutting o Replacement of pre mRNA by RNA o Evolution of RNAs that direct protein synthesis o Evolution of new enzymes that replicate DNA and make RNA copies from it C elegans experiments key to uncovering RNA directed gene regulation o Double stranded RNA can trigger silencing of genes of RNA interference matching sequence microRNAS o genes encode short RNA and control stability and translation of mRNA RISC RNA Induced Silencing Complex Seed sequence match o Silencing RISC released then rapid mRNA degradation mRNA can be degraded once cap and poly A tail Double stranded RNA dsRNA can move between cells and cause
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