BIOL 3333 1st EditionExam # 4 Study Guide Lectures: 28 - 35Lecture 28 (04/06)Applications of Bacterial Synthesis and Propagation (‘Cloning’) and Recombinant DNA (Chapter 9)Purifies Enzymes: an enzyme that is selectively removed from the rest of the cell to get a solution of only one molecule: the enzyme- These are taken from microorganisms and can be used as molecular scissors to break up DNA molecules and then piece them togetherAutomomously replicaiting genetic elements in microorganisms (e.g plasmids) can be used to propagate that DNA within individual cellsRestriction Endonucleases: have a specific recognition sequence (4-6 bp) - many enzymes have palindromic recognition sites- DNA cut within these enzymes is broken w/ in the strand endonulease- some of the enzymes leave a single strand overhang (sticky end) *Bacterial Plasmids have been engineered to serve as vehicles (vectors) for the replication of DNA Important properties: - origin of replication- restriction sites induce DNA- means of selection for inducing plasmid into bacterial cell When you cut a circular plasmid with a restriction enzyme, it yields a linear molecule with “sticky ends”How do you linarize plasmids? - by using a restriction enzymeWhat represents the genomic library? - the entire population of transformantsWhat could a human “library” representing the entire genome represent? - 106 individual coloniesGenomic “library” construction - Digest foreign DNA with the same enzyme- if base 6 recognition site is random in the genome: then it cuts every 4096 bp- human genome = 3 x 109 bp -> 7 x 105 fragmentsLecture 29 (04/08)Viruses: can be used to clone foreign DNA molecules via their ability to propagate in bacteria*Creating a genomic library using phage vectors: - individual phage pick up single piece of genome- infect a bacterial cell with a recombinant phage - MOI = 1- mix with uninfected bacteria plate- all phage plaques taken together represent genomic libraryCosmids: vectors that are hybrids between plasmids and lambda phagecan replicate in a cell like a plasmid or be packaged like a viruscan carry larger DNA inserts than plasmidsDifferent vectors are engineered for different purposes:- plasmids: up to 15 kb- phage: up to 25 kb- Cosmids: 30-45 kb- BAC: 100-500 kb-YAC: 250-2000 kbWhat do classical genetic studies show? - gene A has an interesting phenotypeHow is a Genomic Library constructed?- Biological process of a gene goes under study- the Gene is identified using classical genetics- DNA is the extracted from the organism- The vector is then extracted from bacteria- open up vector - cut DNA fragments- make recombinant DNA - introduce bacteria to cloneInformation on protein sequence can lead to the synthesis of short oligonucleotide probes Screening using related sequences:- if a tissue is enriched in a particular mRNA the mRNA could be used as a probe - an example of this is globin gene sequences- sometimes it is easier to isolate a sequence from one organism than another - the related gene can be used to fish out the same gene from other genomes- an example of this is actin gene sequencesSime mold -> drosophila -> humanGene selection can also be used in screening:- gene must be introduced into a vector so that the information encoded within it can be expressed - if the gene can complement a mutation in the cell into which it is being transformed, then that is a powerful means of isolating the gene of interest - only the clone of interest would survive- if the gene can produce a protein in the cell, then production of that protein product can identify clone Lecture 30 (04/10)Construction of a cDNA library - provides a “snapshot” of the genes expressed in a particular tissue-isolate mRNA from tissue of interest-convert to d.s. DNA in vitro with reverse transcriptase and Pol1-prepare ends for cloning (remove hairpins, add ligate on restriction sites)-clone into vector and propagate in microorganism Polymerase Chain Reaction:Allows for amplification (essentially cloning) of small amounts of DNA from biological samples- provide specific primers flanking region of interestKary Mullis - procedure can be automated using DNA- poymerases from thermophillic organismsDNA sequencing: the chain termination method - dideoxynucleotides used to stop synthesis at a specific baseIn this: 4 different DNA synthesis reactions are runbegin synthesis from specific priming pointadd components for DNA synthesis + a specific ddntp for each of the four reactions Fragments of a discrete size accumulate in each reaction - run the 4 differentreactions electrophretic gel - smallest fragments (closest to primer) migrate farthestLecture 31 (04/13)Automated DNA sequencing: - Each exaction can be specifically labeled with a fluorescently tagged molecule - since each terminated fragment is tagged with a different color reactions can be conducted and run on the same lane- a laster is built into the gel apparatus - this is used to illuminate the bands and a spectrophotometer can record the wavelength and intensity of the fluorescent signal as the DNA band passes the detectorAutomated DNA sequencing:- each reaction can be specifically labeled with a fluorescently tagged molecule- since each terminated fragment is tagged w/ a different color, all reactions can be conducted and run on the same lane- a laser built into the gel apparatus can illuminate the bands and a spectrophotometer can record the wavelength and intensity of the fluorescent signal as the DNA band passes the detector*the output of the detector can be sent directly to a computer and the DNA sequence recorded as the gel is being runNext generation sequencing: - are able to produce sequence data in much larger volumes and at much lower costs- will allow individual sequence info to be collected much more easily- cost of human genome project: 2.7 billion- goal of next gen. sequencing: the 1000 genome- 5/12 sequencing costs using next generation technologies: around 9500/genome- 6500 todayMassively Parallel Sequencing Emulsion PCR: - a single DNA fragment is amplified in a “mocroreactor”- water in oil emulsion generates millions of micelles- each micelle contains all reagents/templates for a PCR reactionPyrosequencing:- cycle nucleotides one base at a time- template and dNTP polymerase = template - DNMP + PPiEnzyme beads: -DNA polymerase: adds nucleotide (dNTP)-Pyroposphate is released (PPi)-Sylfurylase creates ATP from PPi and APS-Luciferase hydrlyses ATP