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BIMM 100 Lecture 7

BIMM 100 Lecture 7 - BIMM100: Lecture 7 ­ Finding...

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Unformatted text preview: BIMM100: Lecture 7 ­ Finding genes and using clones Read: 198 ­204 Also, classic experiment (on PCR) Recombinant DNA •  Overview and geneMc concepts •  Cloning –  Tools •  RestricMon endonucleases •  Ligase •  Polylnkers (MCS) –  Building a clone •  Libraries –  A source of clones –  Screening •  •  •  •  A beXer explanaMon of gel electrophoresis Brief review, and discussion of Defining what you’ve cloned cloning with PCR PCR Learning from hybridizaMon: blots, in situs, etc Expression cloning The visualizaMon (and interpretaMon) of gel electrophoresis M 1 2 3 4 5 6 M x x 8 x x x 2 * * * * * x x x x x x 1 x 3 * = vector x = inserts * x x The experiment: a restricMon digest! You have subcloned gene X into a 3kb vector using the EcoRI endonuclease. Then, you cut these clones with EcoRI and ran them on a gel. How many clones are unique? Are there idenMcal clones? Clone 5 EcoRI gene M 1 2 3 4 5 6 M x x 8 x x x 2 * * * * * x x x x x x 1 EcoRI EcoRI O R I 2.5kb 8kb x 3 * x x ampr * = vector x = inserts Vector piece: 3kb 3 EcoRI sites. 1 is internal (inside your GOI) What is the total size of this plasmid? Clone 6 EcoRI EcoRI EcoRI gene M 1 2 3 4 5 6 M x x 8 2kb x x x 2 * * * * * x x x x x x 1 EcoRI O R I 2.5kb 8kb x 3 * x x ampr * = vector x = inserts Vector piece: 3kb 4 EcoRI sites. 2 are internal (inside your GOI) What is the total size of this plasmid? Recombinant DNA •  Overview and geneMc concepts •  Cloning –  Tools •  RestricMon endonucleases •  Ligase •  Polylnkers (MCS) –  Building a clone •  Libraries –  A source of clones –  Screening •  •  •  •  Brief review, and discussion of Defining what you’ve cloned cloning with PCR PCR Learning from hybridizaMon: blots, in situs, etc Expression cloning Sequencing: the animaMon What do you need? Template Primer dNTPs Labeled ddNTPs DNA pol Check out the animaMons! What if you have a piece of DNA that is too big for one sequencing reacMon? primers Regions sequenced Design new primers! Regions sequenced Known as “walking”: successively building sequence on the basis of new informaMon Other strategy? Shotgun cloning ­ covering the genome with lots of sequencing reacMons and then assembling them by a computer! PCR: what you need •  Template (target DNA) •  SyntheMc oligonucleoMde primers (with homology to the ends of the targeted sequence) –  Length? About 20nts (420 ~ 1012) should ensure they’re specific –  Base composiMon? 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 reacMon (thermocycler) PCR ­ review the animaMon! Important ­ will probably answer some of your conceptual quesMons about PCR! Using PCR to clone genes: an alternaMve to restricMon endonuclease digesMon BamHI HindIII Design primers to flanking edges of your GOI. The primers should have 20bp of complementary sequence. Then add on a restricMon site! It won’t anneal to the DNA, but you can use it later for inserMng the piece into a vector! Why use two different restricMon endonucleases? PCR cloning conMnued PCRing allows you to generate a lot of insert for your reacMons. 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 sensiMve! Working with clones and sequences •  Southern blot –  Detects DNA fragments in genomic samples •  Northern blot –  Detects specific RNA fragments •  In situ (in posiMon) hybridizaMon –  Detects DNA or RNA in specific locaMons •  Microarrays –  Gene expression ManipulaMng DNA and RNA: hybridizaMon Remember back to Lecture 1: the ability of DNA and RNA to be denatured, and then pair specifically with its complement in complex mixtures is tantamount! ApplicaMons: PCR, Southern, Northern, in situs, microarrays What kind of quesMons can you answer with these assays? •  Southern Blot: DNA hybridizaMon –  Is your GOI there? Any change in diseased vs. healthy organisms/Mssues/cells/etc? •  Northern Blot: RNA hybridizaMon –  Is your GOI expressed? Change in diseased vs. WT? •  In situ: DNA or RNA hybridizaMon –  Where is gene expressed (RNA) –  Where is gene located (DNA) •  Microarrays: DNA hybridizaMon –  What are the paXerns of gene expression? Southern bloqng •  Uses restricMon enzymes and gel electrophoresis to isolate, probe for, and detect a parMcular DNA sequence •  Steps? RestricMon digest of DNA Separate pieces by electrophoresis Place gel in NaOH to denature DNA Transfer DNA onto filter Allow labeled DNA probe to hybridize to its complimentary DNA strand –  Wash away the probe –  Expose to x ­ray film. The dark bands on the film are DNA fragments to which the probe has hybridized –  –  –  –  –  Southern Blot: finding a needle in a haystack Need controls! Compare your sample of interest to genomic DNA, subclones, other species, wt samples, etc. Can idenMfy mutaMons, rearrangements, polymorphisms, etc. M 1 2 3 4 5 6 Major use? Does your Sample 2 lacks a band ­ is transgenic animal have gene missing? Does the the gene you tried to probe not recognize it? Is insert (or remove) from it mutated? it’s genome? Northern Bloqng •  Same process as a Southern. Difference? Now we are probing for complementary RNA sequence. Note: remember that RNA has secondary structure (even single strands). This needs to be denatured! •  Probe can be DNA or RNA •  Oren used to determine expression of genes transcribed under certain condiMons (diseases, arer acMvaMon of some process, etc.) by probing for their transcripts. •  Old school! •  New school? QuanMtaMve PCR or microarrays Northern blots: specific RNA detecMon Use Northerns to detect the expression of specific genes Material on blot? RNA Your probe? Your GOI (DNA or RNA) At ler? InducMon of MEL cells that arer drug treatment will differenMate into RBCs. This is measured by β globin, a specific RBC transcript. Key: not only can you tell if the gene is expressed, you can also tell how much is expressed (by the density of the band)! Northerns: more informaMon •  Different blots for different experiments! –  Tissue specific blots: compare expression in brain, liver, thymus, skin, etc. –  “Disease” blots: compare wt cells or Mssues to diseased cells or Mssues –  Pan ­species blots: is the gene expressed in corn? Yeast? Bacteria? Zebrafish? Mice? QuanMtaMve PCR (qPCR or qRT ­PCR): the new school way to measure transcript levels Products that come up sooner are more abundant! Isolate RNA from cells/Mssue/etc Perform RT reacMon (to make cDNA) Add primers, Taq, and fluorescent dye that will bind to double stranded DNA Perform thermo cycling in special machine that records fluorescence arer every cycle Correlate the amounts of product Controls are important ­ usually you normalize GOI expression to a “housekeeping transcript” Northerns vs. Southerns •  Northerns tell us expression of a gene •  Southerns tell us if the sequence is in the genome •  Key: the posi,onal informaMon of these genes and transcripts are lost. –  How can we see where a gene is located? –  How can we see where a transcript is located? Easy! In situ (in posiMon or in place) hybridizaMon! •  Two different types –  DNA: look for locaMon of genes in chromosomes •  Why? TranslocaMons, inserMons, deleMons, etc. –  RNA: look for where genes are being expressed •  Why? LocaMon of gene expression, changes over developmental Mme windows, etc. •  Can use different probes –  Fluorescent (FISH) –  enzymaMc DNA FISH: molecular diagnosMcs Green: BCR (chromosome 22). FuncMon not enMrely clear ­ has kinase acMvity Red: ABL (chromosome 9). Important for cell division, differenMaMon, cell cycle control, etc. Bcr/abl rearrangement in chronic myelogenous leukemia (aka the Philadelphia chromosome). Where red and green are next to each other indicates the fusion. MitoMc cells Lyse and fix Denature DNA Hybridize with probes Visualize RNA in situ hybridizaMons enzymaMc Detects temporal and spaMal gene acMvity Use nucleoMdes to find RNA in whole animals, secMons, cell preparaMons, etc. In situ: an actual experiment Does gcsf mRNA injecMon increase HSCs? Animals were injected at single cell stage, fixed, processed, and probed at 36 hours. gcsf injected Mock injected Probed with cmyb (a gene expressed by HSCs) What’s the conclusion? What are the controls in this experiment? What are some other experiments that could be done? Other methods of gene expression: microarrays Key: Comparison of gene expression between two samples An experiment: comparing gene expression and how it correlates with nutriMonal state Two condiMons: Serum No serum Prepare cDNA from both (RT!). Label the cDNA with green ( ­serum) or red (+serum) Hybridize to array, and wash away non ­ bound fragments Measure green and red fluorescence at each spot (different sequences of DNA) How to read the actual array data Green: condiMon 1 (no serum) ­ change in gene expression in that sample Red: condiMon 2 (+ serum) ­ change in gene expression Yellow: overlap ­ expression in both samples (no change in gene expression) Black: no (or really low) expression ­that gene is not expressed Working with the data: heat maps (not the actual array) 500 out of 8600 genes change their expression paXern in presence of serum over Mme! •  •  •  •  Each column? A single gene. Each row? A different Mme point. Computer can cluster genes either overexpressed (red), under expressed (green), or not altered (black) in comparison to a control ­ CRITICAL! “Trees” on top show clustering of related genes (the computer is programed to do this ­ it doesn’t know what is related ­ you tell it what is related!) Ex. Cell ­cycle control genes might all be clustered… Changing the scale: microarrays •  Can now look at thousands and thousands of genes at a Mme •  Technology is constantly being refined and further developed –  Ex. Most arrays use 60bp instead of 20bp oligos now. Why? –  Geqng cheaper, more efficient, and more standardized (i.e. what are good controls? What should the computer values be set at? Lots of issues!) •  Will be essenMal in the future for diagnosMcs –  Is a tumor gene being expressed? –  Does that gene correlate with a certain prognosis? –  Is a different treatment modality warranted? How else can we study our cloned genes? •  Make protein from the cDNA –  General strategies and consideraMons –  PotenMal problems •  What to make? •  How to make it? •  How to purify it? •  How to test it? Medical use? Using bacteria (and other cells) as factories for medically useful compounds •  A case study: granulocyte colony sMmulaMng factor (gcsf). –  Murine GCSF was first purified in 1983. –  Human GCSF was discovered and purified in 1985. –  Binds to its cognate receptor (GCSFR) and induces downstream intracellular signaling through JAK/STAT/ SOCS pathway. –  Biological acMviMes: •  Produced by monocytes, fibroblasts, and endothelial cells •  Up ­regulated in emergency situaMons, such as LPS infecMon •  Expands neutrophils and monocytes, mobilizes HSCs Loss of GCSF signaling results in serious clinical manifestaMons •  GCSF ­/ ­ and GCSFR ­/ ­ mice: –  Severe neutropenia (lack of granulocytes and macrophages) and reduced neutrophil survival –  DefecMve granulocyte macrophage progenitor (GMP) repopulaMon and differenMaMon. •  GCSF is uMlized in the clinic: –  To mobilize hematopoieMc progenitors from the bone marrow (mechanism is sMll unknown) –  To treat neutropenia induced by severe infecMon, radiaMon therapy, chemical exposure, or congenital defects in neutrophil maturaMon. •  Neupogen (filgrasMm) (Amgen): recombinant human GCSF •  Neulasta (pegfilgrasMm) (Amgen): more stable, longer lasMng version of recombinant GCSF All due to recombinant DNA technology! Expressing a cloned cDNA in bacterial cells •  Classic inducible protein producMon method –  In the presence of IPTG (a lactose analog), RNA polymerase transcribes the lacZ gene, which is translated into β ­galactosidase –  This is under the control of the lac promoter •  Different promoters/inducers can allow experimental control of expression Expressing a cloned cDNA “Trick” the bacteria by puqng your GOI (gcsf here) under control of the lac promoter, instead of lacZ. Called subcloning. Adding IPTG now forces the cell to generate your GOI! Now you need to purify the protein. Hint? Add a “tag” to the protein… Success stories ­ recombinant technology to the rescue Product Medial purpose RestricMon endonucleases More cloning! Cloqng factors (VIII & IX) Hemophilia A & B ErythropoieMn (epo) Anemia Insulin Diabetes (type I) Growth hormones Growth hormone deficiencies Interferons Leukemia, hepaMMs, MS DNAse CysMc fibrosis Some possible issues •  •  •  •  •  Expression (can be toxic to host cells) PurificaMon (someMmes can be very difficult) Immunogenic Inappropriate uses (especially epo and gh) Not fully funcMonal –  May need processing, modificaMon, glycosylaMon, and special help folding not possible in bacteria Check your understanding! •  Compare: –  Southern Blot and Northern analyses –  Microarrays and in situs •  What material is being analyzed? •  What is the probe? •  What will you learn from the assay? •  What controls do you need? –  Expression cloning •  What do you need? •  What are some problems? Next class: •  Expression cloning –  In bacteria •  What do you need? •  What are some problems? –  In animal cells •  What do you need? •  What are some different expression techniques? •  What are some issues? ...
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