BIO 305 1st EditionExam 3 Study Guide: Lectures 15 - 21Lecture 15 – Genetic Information & DNA StructureWhat three aspects define genetic information? Is DNA the only source of genetic information?Griffith ExperimentAvery, Macleod ExperimentHershey-Chase Experiment – What does semi-conservative replication look like? What about conservative or dispersive replication?Nucleotide StructureWhat components make up a nucleotide? What is the difference between a nucleotide and nucleoside? What part is the DNA backbone? What is the structural difference between DNA and RNA? What is a ddNTP? What is the advantage of having uracil as an analog to thymine? Which bases are purines? Which are pyrimidines?What are Chargaff’s rules? What conditions of DNA lead to these rules?DNA StructureWhat are the important features of the DNA helix? What are the purposes of a major and minorgroove? What are supercoils, why and how are they formed? What is the difference between positive and negative supercoiling? Does genome size determine organism complexity? What are the impacts of DNA structure on nature? What some discoveries facilitated by DNA structure? Is DNA as reactive as RNA?Lecture 16 - Methodsa DNA  mRNA  ProteinHow do we study genes and gene products?1) Have to be able to section and single about specific fragments2) Have to be able to read RNA involved3) Have to be able to analyze productsGenomic DNA Analysis Flow Chart – understand which situation you would use each method in and how to interpret each methodGenomic DNA  restriction digest  vectors (pick plasmids, fosmid, BAC, YAC, or bacteriophage)  bacterial clones  amplification and recovery  autoradiogram with a DNA probeGenomic DNA  restriction digest  gel electrophoresis  Restriction Mapping (with double restrictions)Genomic DNA  primer building  PCR  Southern Blot  autoradiogram or Sanger Sequencing or High Through put SequencingDNA  genomic DNA library  select a clone  Southern Blot  autoradiogrammRNA – tells you about expression or splicing:RNA  Northern Blot  autoradiodiagramRNA  reverse-transcription to make cDNA  cDNA library  Southern Blotor  PCR  Southern Blot/Sanger/High Through putProtein Protein  gel electrophoresis or Western Blot or Autoradiogram with Antibody ProbeMore on Methods:Generating genomic libraries with 95% coverage:# clones needed = (probability of restriction enzyme site)*(length of genome)*# of equivalentsPCR – List all the steps: Denaturation, Annealing, Elongation. Why is restriction not required? What are the typical key reagents of PCR? What do each do? How are primers built, how are they used to? Why are some fragments linearly amplified while the desired fragment is exponentially amplified? How does reverse transcriptase work and when do you use it for PCR?Sanger: What reagents do you need for Sanger sequencing and how do they work?To read a Sanger sequences, starting at the top of the gel to the bottom, write out the COMPLEMENT of the bases. This will give you the original DNA sequence from 5’  3’If genes are close to each other, need two primers to express anything in between. The final product of amplification of DNA using PCR will include the primers! You want to pick the 5’  3’ of the top strand as the left primer and the 5’  3’ of right primer will be the complement of the right end from 3’  5’ (sense and anti-sense)High-through put Sequencing: 1) DNA is extracted and fragmented to produce a ‘library’ (a largepool of short sequences) 2) Align them to reference sequences to obtain whole genome sequenceNextGen sequencingLibrary preparation – performs massive parallel sequencing, adenylation of sheared ends, adenylose are attached, selected and purifiedCluster system – dense lawn of aligose hybridize covalently to the single-stranded fragments, reverse strands are washed, and ends are ‘capped’Sequencing – simultaneous base-by-base synthesis, lasers excite and indicate newly synthesized DNA, allows for identification of difficult sequencingHow do you conduct identity testing using STRs?Lecture 17 - ReplicationDNA (+replication)  transcription  RNA  translation  protein (+ prions) ( reverse transcription) (DNA  transcription factors)The Meselson-Stahl Experiment: What does semi-conservative replication look like after one round of replication? Two rounds? What about conservative or dispersive replication?Label the 5’ and 3’ ends of a replication fork. Be able to solve for the rate of a replication, given length of genome, given number of ORIs with given information.#ORIs = (length of entire fragment)*time given/(b per minute) 2 (because ORI has two moving forks)Which strand is the template strand, non-coding strand, or coding strand?Which is the lagging strand? The leading strand? Be able to label the 3’ and 5’ ends and describe how their processes are different.How is replication initiated, elongated, and terminated? How do prokaryotes and eukaryotes differ in these steps?The Replisome:Note: DNA Polymerase does NOT produce Primers; this is done by RNA polymeraseNotice that exonuclease can work both ways: proofreading is 3’  5’!DNA Pol I does primer removal and filling of gaps, DNA Pol II does DNA repair, DNA Pol III does DNA synthesisStarts with Helicase, DNA Pol III dimer works toward Helicase, RNA primer and primase works in opposite direction. The definition of the 3’ strand and the 5’ strand depend on which proteins are boundto itProkaryote Replication (Left) VS. Eukaryote Replication (Right)How is termination complicated by the lagging strand? What are telomeres and what do they do? Describe the structure telomerase and how it works? What would happen without it? Where does it work? Be able to label the 5’ and 3’ ends.Lecture 18 - TranscriptionInitiation: How is it initiated and what are the differences between Prokaryotes and Eukaryotes?ProkaryotesPromoter Element : -35 and -10 elementsWhat binds here: sigma factorList of holoenzyme components: sigma, 2 alpha, beta, beta’, omegaEukaryotespromoter elements: TATA boxWhat binds here: TBP – tata binding protein & many transcription factorsElongation: How is it elongated? Why are there many different types of RNA?Eukaryotic RNA Polymerases:RNA Pol I – transcribes ribosomal RNARNA Pol II – transcribes mRNA (has 14 subunits! Two