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Biol 208 Final Exam Notes

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BIOL 309
Kyra Jones

Bio 208 Final Exam Notes Denaturation MethodsLower salt concNegative strands repelIncrease tempBreaks hydrogen bondsIncrease pHChanges charge on hydrogen bondsCrosslinking Base pair with improper reagentsbasesHydrophobic Reduces DNA stabilitysolventsCG bond has 3 H bonds while AT has 2 H bonds betweensinglestranded DNA absorbs UV light more strongly because no longer have stacking interactions between basesRNA is more susceptible to degradation by pHRNA secondary structures aid in mediating tRNA structure transcriptions terminators mRNA stability and controlling gene expressionall DNA strands are extended in the 53 direction requires 3 OH primers while RNA does not need a primerB helixnormal righthanded 1 turn per 10bpA helixlower water content righthanded 1 turn per 11 bpZ helixCG high salt lefthanded irregular structuretopoisomerasesresponsible for supercoiling of DNAProkaryotesEukaryotespolycistronic mRNAeach gene has its own promoterseveral genes controlled by 1 contains intronspromoter called an operonRNA polymerases do not bind to DNA transcription is carried out by RNA directly and require certain transcription polymerasefactorsRNA Ps bind to promoter and recognize a specific site downstream2 recognition sites at 35 and 10ShineDalgarnoribosome binding site in prokaryotes for the initiation of translation steadystate of proteinamount of protein produced from a transcript is a function of the rate of protein synthesis and rate of protein degradationcoding regionORF DNA that encompasses the sequence from the start to stop codon including introns in eukaryotescoding sequence exons actually translated into a proteinmRNA contains 5 and 3 UTR as well as exons Module 1 Studying Biology at a Molecular Levelgenetic approachinitial studies of development or physical processesbiochemical approachanalyze proteins that can be measured in vitrobioinformaticscomputer analysis of nucleic acid and protein sequences as well as accessing the data from genomewide experiments usedGenetic Approachprocesses that cannot be studied in vitroidentification of the causes of these phenotypes What is a mutantHow are mutants created DNA replication mutagens transposonsWhat types of DNA changes might a mutant have substitution insertiondeletion inversiontransition purine to purine AGpyrimidine to pyrimidine replacement CTtransversion purine to pyrimidine and vice versaHow are mutants of interest identified determine what is normal and screen or selectforward genetics find a mutant and identify which gene causes the phenotypereverse genetics start with a gene of interest and create an organism that expresses the gene abnormally Q if another gene copy existed but the coding region was reversed relative to the promoter what would happenA the gene would be untranslated because mRNA gets degraded via gene silencing siRNAs or miRNAs1isolate gene altered in the mutant check databases2isolate corresponding cDNA sequence3determine DNA sequence of cDNA check databases4clone into a vector for overexpression5purify protein and characterize check databsesBiochemical Approachexample of DNA ligasereveals its kinetics and protein characteristicsmust prepare an assay mix containing the protein of interest in its active state ideal environmentmust be able to measure the protein quantitatively with respect to time or protein concentrationoften necessary to purify the protein from a mix using chromatography1purify the protein to homogeneity2determine amino acid sequence check database3create an oligonucleotide probe4screen genomic or cDNA library5sequence isolated gene check databaseBioinformaticscombination of biology and computer science that involves the collection of protein and nucleic acid info Into searchable databases that allow comparative analyses to be simplifieddata could revealrelated genes similar genes in other organisms mRNA sequences map position start site of transcription site of mutation relative to wildtypeif studying a particular enzyme activitycan find the known sequence of a related enzyme the presence of cDNA sequences encoding this enzyme based on a known amino acid sequence indicates presence of other members in the enzyme pathwayModel Organismssimilar features to those of more complex organismssmall genome size often diploid low amount of repetitive DNA small and cheap to maintain short life cycle transformableadvantagescost effective collaborative fewer organisms share resources outside perspective validate results share data Module 2 How to Clone a Genecloning asexual propagation of genetically identical organisms1DNA is fragmented with restriction enzymes2vector is linearized with the same restriction enzymes3they are ligated together and transformed into the hostLibraries and Hybridizationligate each of the restriction fragments of a genomic digest into a vector and each bacterial colony is transformed with a different recombinant fragmenttransformants are screened with a probe hybridization hybridization reaction requiresdenatured DNA template primer dNTPs some labeled buffer with MgCl2 required by DNA polymerase DNA polymeraseGenomic CloningPCRcouple of weeksonly a few hoursneed a probeneed sufficient info to design primersinvolves radioactivityno radioactivity neededModule 4 DNA Purification and Fragment IsolationExtraction and Purification of Nucleic Acids1collect tissue containing the nucleic acid of interestfresh material quickly frozen if unable to2homogenize tissue and lyse cells blender sonication thaw liquid homogenization manual grinding enzymes detergents solventsEDTA ethylene diaminetracetic acidholds metal ions to prevent DNAses from damaging DNASDS sodium dodecyl sulphatedissolves cell membrane denatures proteins inside the cellLysing MethodsBacterialiquid homogenization forces cell suspension through a narrow spacesonication sound waves form imploding bubblesAnimalsfreeze then thaw ice crystals contractPlantsfreeze with liquid nitrogen grind with mortar and pestleYeastglass beads crush cell wall micropipettors often cause shearing of the chromosomal DNA while plasmids are
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