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BIMM 100 Lecture 9

BIMM 100 Lecture 9 - BIMM100 Lecture 9: wrap up and...

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Unformatted text preview: BIMM100 Lecture 9: wrap up and review of molecular biology techniques Office hours: 3 ­4 pm today SecFons: regularly scheduled today, cancelled on Tuesday and Wednesday Exam tomorrow! •  Exam will be in class (8 ­9:20am). If you’re late, you just lose Fme ­ exams must be collected at 9:20, as there is a class right aTer ours! •  Bring your student ID, and a pen. •  Please: –  No hats –  No cell phones (or silence them and have them in your bag or pockets) –  No papers or books on your desks. –  Don’t use white out! –  Don’t use a pencil ­ you cannot have your test re ­graded if there is an issue! What should I know for the test? •  Chapters 4 & 5 –  Key concepts •  QuesFons at the end of each of these chapters –  Key terms –  Review the concepts –  Analyze the data •  Classic experiments What should I know about the classic experiments? •  A review: the scienFfic method! –  “A method of procedure that has characterized natural science since the 17th century, consisFng in systemaFc observaFon, measurement, and experiment, and the formulaFon, tesFng, and modificaFon of hypotheses.” Oxford English dicFonary –  Steps of the scienFfic method: •  •  •  •  •  Ask quesFon Construct hypothesis Test with an experiment Analyze results Form conclusion Example: Reverse transcriptase discovery •  The observa+on: Temin, 1961 ­ discovered that RSV was sensiFve to inhibitors of DNA synthesis. And, cells had viral DNA that was integrated into their genome. How was this possible? •  Another observa+on: BalFmore ­ isolated an RNA ­dependent RNA polymerase acFvity in RNA viruses. Why? Example: Reverse transcriptase discovery •  The ques+on: Can a retrovirus perform DNA synthesis? How can this be tested? •  The experiment: –  Step 1: disrupt viruses –  Step 2: add radiolabeled dTTP, normal dATP, dCTP and dGTP to the viral prep. –  Step 3: measured radiolabeled dTTP gekng incorporated into nucleic acid. CONCLUSION? DNA SYNTHESIS WAS OCCURRING! Example: Reverse transcriptase discovery •  The ques+on: Can a retrovirus perform RNA synthesis? How can this be tested? •  The experiment: –  Step 1: disrupt viruses –  Step 2: add radiolabeled rNTP to the viral prep. –  Step 3: measured no radiolabeled RNA being synthesized. CONCLUSION? RNA SYNTHESIS WAS NOT OCCURRING! Example: Reverse transcriptase discovery •  The ques+on: What was the product being formed? •  The hypothesis: it was RNA (based on the prior experiments) How can this be tested? •  The experiment: –  Step 1: take product –  Step 2: add RNase –  Step 3: measured no destrucFon of the product CONCLUSION? THE PRODUCT WAS NOT RNA! Example: Reverse transcriptase discovery •  The ques+on: What was the product being formed? •  The hypothesis: it was DNA (based on the prior experiments) How can this be tested? •  The experiment: –  Step 1: take product –  Step 2: add DNase –  Step 3: measured destrucFon of the product CONCLUSION? THE PRODUCT WAS LIKELY DNA! The data that showed this? Standard condiFons? Try to make DNA with normal condiFons. Rnase treated? Now, less incorporaFon of radioisotopes (i.e. the product was not being formed)! This implies that RNA is essen+al for this ac+vity (remember that the genome of these viruses is RNA! What is the negaFve control here? The data that showed this? Untreated product? What is the product? Rnase treatment of the product doesn’t do anything. But, Dnase treatment reduces the amount of radiaFon incorporated (i.e. is being destroyed) This implies that DNA is is the product being made! What is the negaFve control here? Example: Reverse transcriptase discovery •  So, we know: –  DNA is the product being made –  DNAse doesn’t prevent this acFvity (DNA is not likely the template) –  RNA is NOT the product being made –  RNAse prevents this acFvity (RNA is likely the template) •  Conclusions: –  Retroviruses have an RNA genome that somehow generates a DNA product Example: Reverse transcriptase discovery •  The beauty of this experiment? –  Simplicity! –  Controls! –  Importance! •  1975 nobel prize awarded to both BalFmore and Temin for this work •  Has been essenFal for generaFng cDNA libraries, ways to do microarrays, etc. •  Has been essenFal for understanding retroviruses and their role in human diseases! Some scienFfic Fps? •  Design every experiment so that they will have a clear answer. •  Design experiments in such a way that you are only altering one variable at a Fme (make sure they are well controlled!). Some review! •  PCR and it’s uses! •  Common molecular biology assays! •  More on knocking out genes! Polymerase chain reacFon (PCR) •  What’s it good for? –  Making probes –  Making genes for cloning (from cDNA) –  GeneraFng pieces of genomic DNA (from genomic DNA) –  “tagging” genes by inserFonal mutaFons (don’t worry about this for the exam ­ it’s basically an easier method of performing funcFonal complementaFon! PCR: what you need •  Template (target DNA) •  SyntheFc oligonucleoFde primers (with homology to the ends of the targeted sequence) –  Length? About 20nts (420 ~ 1012) should ensure they’re specific –  Base composiFon? Try to have primers = G/C numbers (equal Tm) –  Perfect match (can also do near matches) •  dNTPs •  Thermostable DNA polymerase (Taq, from Thermus aqua,cus, a bacteria that lives in hot springs) •  Means of controlling the reacFon (thermocycler) Great use of PCR: to generate genes for cloning! BamHI HindIII Design primers to flanking edges of your GOI. The primers should have 20bp of complementary sequence. Then add on a restricFon site! It won’t anneal to the DNA, but you can use it later for inserFng the piece into a vector! Why use two different restricFon endonucleases? PCR cloning conFnued PCRing allows you to generate a lot of insert for your reacFons. Easy and fast! But, PCR does have one downside ­ it makes mistakes every once in a while (if you are using a non ­proofreading polymerase). How can you make sure that the DNA piece that you just cloned is correct? Two ways ­ one is more sensiFve! Your common methods: know them! Assay What does it tell you? Southern blot Presence or absence DNA of genes, size of genes Labeled RNA or DNA Northern blot Gene expression level, size of transcript Labeled RNA or DNA In situs Gene expression in RNA or DNA temporal and spaFal manner (RNA) or gene locaFon (DNA) Labeled RNA or DNA (respecFvely) Microarray Gene expression cDNA differences between two samples DNA oligos immobilized on a slide QuanFtaFve RT ­PCR Gene expression level What is being Probe analyzed? RNA cDNA N/A ­ detected by DNA dye that only binds to dsDNA Some things you need to know: the inacFvaFon of genes •  BIG quesFons! –  Is a specific gene essenFal for viability? –  What is the funcFon of a gene within the cell? –  What role to genes play in the development and maintenance of the whole organism? How can we disrupt genes? •  The expression of a gene can be decreased (or eliminated) by… –  Genomic disrupFon of the gene of interest •  Yeast •  Mice –  Global (ubiquitous KO) –  CondiFonal KO –  Tissue specific KO –  Post transcripFonal gene disrupFon •  RNAi •  morpholinos Gene inacFvaFon in yeast •  Gene inacFvaFon in yeast (S. cerevisiae) 1.  Make disrupFon construct 2.  Introduce it into yeast cells 3.  Select for cells that integrated the disrupFon construct 4.  Study the cells with the deleted gene of interest Gene inacFvaFon (in yeast) Step 1: building a disrupFon construct Make primers with sequence homologous to <20nt to the DNA sequence on either side of the GOI, as well as to kanMX You need at least 20nts! kanMX confers resistance to G418 in yeast Construct with kanMX and homologous sequence ends Gene inacFvaFon (in yeast) Homologous recombinaFon will allow the integraFon of the disrupFon construct into the genome. KEY? The homologous sequence ends… Heterozygous mutant Select for cells that have undergone recombinaFon by adding G418 This is a great test to see if a gene is essenFal for viability! From yeast to mice (and other mammals) Steps: 1.  Make a targeFng construct 2.  Introduce it into murine ES cells 3.  Select for desired embryonic stem cells –  PosiFve selecFon: Select for cells that incorporate the targeFng construct –  NegaFve selecFon: Select against the cells that incorporated the targeFng construct by random integraFon 4.  Generate mice in which the gene of interest is disrupted in every cell (ubiquitous KO) 5.  Study the effect of the gene disrupFon on the development and maintenance of a whole organism (the mouse in this case). Making a targeFng construct A B C Gene X genomic DNA Exons A neo C tk Gene x targeFng construct Regions of homology Neomycin: posiFve selecFon ­ confers resistance to G418 Thymidine kinase (tk): negaFve selecFon ­ confers sensiFvity to ganciclovir. Cells normally have tk, but this gene is from the herpes virus, which inhibits DNA replica,on in ES cells. IntroducFon of targeFng construct into murine ES cells A neo C tk TransfecFon, electroporaFon, etc A B C A A B B C C SelecFon of cells that incorporate the targeFng construct Homologous recombinaFon: occurs at a very low frequency. That’s why you need to add a selectable marker! KEY: only things that are between the homologous regions are integrated! Things outside of the regions of homology are not! SelecFon against cells that incorporate the targeFng construct by random integraFon Random inser+on: occurs at a very high frequency. That’s why you need to add a selectable marker to prevent this! KEY: Random inserFons include the whole construct! Not just homologous regions! Why do we not want random inserFons? PosiFve and negaFve selecFon ­key steps in this process! Now we have targeted disrupFon of a gene in ES cells. What’s next? •  For ease, we will choose a simple method to detect the chimeric animals that we will create. –  Chimera: an animal with Fssues derived from transplanted ES cells and host cells. •  Easiest method? Coat color! What do we have? •  Targeted embryonic cells from a brown mouse heterozygous for our gene x (x+/x ­). Since we started with ES cells from a brown mouse, and this coat color is a dominant allele, the ES cells are also (A/A) What do we need? •  An early embryo capable of incorporaFng ES cells –  Choose a recipient that has a different marker allele (lets say black hair ­ the genotype of this mouse is x+x+ (because it is wildtype for gene x) and a/a (because it does not have the dominant brown fur color). –  RecepFve pseudopregnant mouse to serve as a “surrogate mother” •  Done with hormones, or by maFng with a “fixed” male IntroducFon of mutant cells into mice Remember: A is brown coat a is black coat You can use any selectable marker ­ coat color is just so easy to visualize! GeneraFon of mice with ubiquitous disrupFon of gene x Remember: you need to get integra+on in germ cells, or else these genes will not get passed on to future generaFons! You are only interested in mice that are brown (i.e. are derived from the targeted ES cells) GeneraFon of mice with ubiquitous disrupFon of gene x Now, you can look at phenotypes to find a funcFon! Why would you want a condiFonal KO, versus a ubiquitous one? •  What if the KO mouse is inviable? •  What if a gene is important for development? •  What if you want to isolate the funcFon of a gene in a parFcular Fssue? So, we need a way to modulate the expression of genes in different Fssues One example of an inducible system: Cre loxP •  A recombinaFon system derived from bacteriophage P1 that also funcFons in mouse cells Introduced by non ­ homologous recombinaFon loxP: site specific DNA recombinaFon sites Cre: enzyme that catalyzes recombinaFon between LoxP sites : Fssue specific promoter you use to direct Cre expression The nuts and bolts of the cre loxP system recombinaFon Remember: you don’t need to control gene expression at the transcripFonal level! •  You can use post ­transcripFonal modulaFon of genes! –  One example? RNAi (RNA interference) ­ using dsRNA to degrade a parFcular mRNA transcript. Organisms have devised ways to get rid of what they think may be viral (i.e. bad) dsRNA that may get into their cells ­ it’s a primiFve defense mechanism! RNAi strategy 1.  dsRNA is recognized by cells 2.  dsRNA is processed by Dicer (an RNA endonuclease) found in all metazoans, but not simpler organisms such as yeast into 23nt ds segments called small inhibitory RNAs (siRNAs) 3.  siRNA associate with the RISC complex, and are separated into single strands 4.  These single stranded siRNAs recognize complementary mRNAs and base pair to them 5.  ATer basepairing, the RISC complex cleaves the mRNA/siRNA hybrid molecules Producing specific siRNAs Make the siRNA in vitro with two different constructs 23 nts Producing specific siRNAs Have the cells make dsRNA for you (with an RNA hairpin) Your construct is made of sense and anFsense sequence ­ that’s why it basepairs and makes a hairpin ...
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