BIMM 100 Lecture 6

BIMM 100 Lecture 6 - BIMM100 Lecture 6: using...

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Unformatted text preview: BIMM100 Lecture 6: using molecular biology ­ recombinant DNA techniques & characterizing/ using clones Reading: 186 ­198 Recombinant DNA •  Overview and geneIc concepts •  Cloning –  Tools •  RestricIon endonucleases •  Ligase •  Polylnkers (MCS) –  Building a clone •  Libraries –  A source of clones –  Screening •  •  •  •  Defining what you’ve cloned PCR Learning from hybridizaIon: blots, in situs, etc Expression cloning Libraries and their role in recombinant DNA technology •  A library is a collecIon of clones •  Genomic DNA vs. cDNA (complimentary DNA that represents copies of mature mRNA in DNA form). –  cDNA lacks noncoding regions (introns) of genes. –  Use polyA tails to obtain and create cDNA library •  Insert size consideraIons/ vector limits –  Plasmids (10 ­20kb max) –  Bacterial arIficial chromosomes (BACs; up to 2Mb [2 million nts]) •  RepresentaIon –  How many clones represent a genome? •  For mammalian studies, 106 ­107 of individual genes. Why? Genes are transcribed at different rates ­ need the low and high expressing ones! –  How do we find the clone/sequence of interest? Oligos (primers): what are they? Where do they come from? DMT: dimethoxyItyl (blocks OH from reacIng) OligonucleoIdes (oligos): chemically synthesis Chemical, non enzymaIc synthesis Synthesis is the opposite of the in vivo reacIon ­ occurs from 3’ ­5’! Important for cloning, sequencing, and PCR! Can make precise sequences or introduce random nts! Depends what your goal is! Making a cDNA library in a plasmid vector Need RT! Isolate mRNA from cells or Issues Separate mRNA (which has polyA tails) by binding them to oligo ­dT sequences (could be immobilized on beads in a column. Add oligo ­dT primer, and then reverse transcriptase. The RT will transcribe mRNA into cDNA Building a cDNA library in a plasmid vector conInued Remove RNA (with alkali condiIons or enzymes) Add poly(dG) tail (to hybridize to later) Single stranded cDNA! Hybridize with an oligo ­dC primer (to be able to generate a double stranded version of cDNA) Building a cDNA library in a plasmid vector conInued Synthesize the complementary strand Double stranded cDNA! Methylate (with an enzyme) the EcoRI sites (or whatever other sites you want to use) to protect them! Building a cDNA library in a plasmid vector conInued Ligate a “linker” onto the ends of the double stranded cDNA Then, cleave with EcoRI to generate sIcky ends (for shujling this cDNA piece into a vector for propagaIon Building a cDNA library in a plasmid vector conInued These colonies are your collecIon of plasmid clones: your cDNA library! What do we do with a library? Finding your clone of interest… The pracIcal issues: You now have a plasmid vector ­ based library in E. coli host cells Need to replicate the colonies onto membranes. Then, lyse the cells (on the membrane). DNA sIcks to the membrane, is denatured, and stays in place! Then, you need to hybridize with a probe (in this case the probes are coupled to some form of radioacIvity, but that is not the only way), wash, detect, and “pick” your colony of interest Looking closer at what’s going on “inside” a colony The pracIcal issues: You have already lysed your cells, and denatured the released plasmid DNA. Now, incubate with labeled DNA, which hybridizes to the complementary DNA stuck on the membrane. A washing step will remove the labeled DNA that doesn’t hybridize to the DNA (that is stuck to the filter). Output? In this case, expose to x ­ray film. Probes: more informaIon •  Key for idenIficaIon and cloning •  A good length? About 20 nucleoIdes –  Specific 20bp sequences will only occur once every 420 Imes. Since most genomes (even the human) are smaller than this number, usually one probe will only come up 1x per genome! •  Could be something that has already been idenIfied –  By comparaIve cloning between similar species, gene families, etc. –  Helps if you know the sequence of an organism’s genome •  Could be synthesized based on known/or inferred properIes –  Unique probes are based on known sequences –  Degenerate probes can be based on protein sequences •  Could be determined based on geneIc approaches –  Based on mutant phenotypes •  Could be based on expression cloning –  Looking for anIbodies, secreted proteins, or based on protein acIviIes An example: cloning a gene based on a geneIc approach (funcIonal complementaIon) For selecIon in yeast Finding a funcIonal protein that complements a recessive mutaIon First order of business ­ need a shujle vector! And, it needs to be able to grow in two different organisms; yeast and bacteria MCS ReplicaIon in E. coli SelecIon in E. coli ReplicaIon in yeast Yeast centrosome: necessary for segregaIon in yeast cells ConInuing the building of a plasmid (shujle) vector genomic library Isoschizomers! Use yeast genomic DNA (not a lot of introns), and the genome is compact enough that the enIre sequence of a gene will be included in a DNA fragment inserted into a vector. To ensure proper coverage of the genome, restricIon pieces are made to be about 10kb (“parIally digested”) Based on the size of the fragments (10kb), and the size of the yeast genome, it is necessary to generate 105 E. coli colonies to assure that each piece of the genome will be represented at least 1x. Finding a clone of interest (by funcIon) The principle: the wild type gene will complement it’s recessive mutant phenotype Now ­ isolate DNA from your colony! Only genes complemenIng the mutant cdc gene will grow Recombinant DNA •  Overview and geneIc concepts •  Cloning –  Tools •  RestricIon endonucleases •  Ligase •  Polylnkers (MCS) –  Building a clone •  Libraries –  A source of clones –  Screening •  •  •  •  Defining what you’ve cloned PCR + expression cloning Learning from hybridizaIon: blots, in situs, etc Expression cloning What’s in your clone? •  Now that you have idenIfied a gene that complements your mutaIon, what do you do? •  Perform restricIon analysis and mapping –  Make more DNA –  How big is your insert? –  Do you have “candidates?” Do your clones match the candidates? –  Sequencing •  Used to be prohibiIvely expensive. Now whole genome sequencing is relaIvely cheap. Gel electrophoresis: a method to separate DNA molecules of different length The steps!  ­1. Isolate your DNA (from clone or where ever) 0. Digest your DNA with endonucleases. 1.  Make a polyacrylamide gel (for really small pieces 10 ­2000 nts long) or agarose gel (for pieces from 200 nts to 20 kbs in size). 2.  Add your DNA to the well of the gel. 3.  Turn on the electricity! (Make sure you have the current flowing the correct way!) I usually think… “run towards red,” assuming that the + electrode and wires are colored red… Gel electrophoresis 4.  Keep an electrical current passing through the gel. The negaIvely charged DNA molecules will migrate through the gel based on their size. Small pieces will migrate faster! Large pieces will migrate slower! WHY? Near neutral pH, DNA molecules carry a large negaIve charge. So, they migrate to the posiIve electrode! Gel electrophoresis Aqer you have run the gel, you need to visualize the DNA. You can do this by incubaIng the gel with a fluorescent dye (like ethidium bromide), or if the DNA is labeled with a radioisotope, you could just expose it to x ­ray film. Large piece of DNA Small piece of DNA EtBr ­ intercalates into DNA strands ­ fluoresces aqer exposure to UV light ­ potent mutagen! The visualizaIon (and interpretaIon) of gel electrophoresis Large pieces of DNA 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 A gel aqer electrophoresis. This gel has been soaked in EtBr, which causes the DNA to fluoresce when exposed to UV light. The experiment: a restricIon 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 idenIcal clones? How many EcoRI sites does Clone 6 have? How many EcoRI sites are inside your cloned sequence (in lane 6)? Small pieces of DNA What’s in your clone? •  Now that you have idenIfied a gene that complements your mutaIon, what do you do? •  Perform restricIon analysis and mapping –  Make more DNA –  How big is your insert? –  Do you have “candidates?” Do your clones match the candidates? Use gel electrophoresis to compare restricIon pajerns! –  Sequencing •  Define unique end points with the Sanger ­dideoxy chain terminaIon technique The key to sequencing? Dideoxyribonucleoside triphosphate (ddNTP) The main difference? H instead of OH. It gets incorporated, but terminates elongaIon at that point (it cannot form a phosphodiester bond with the next nucleoIde). With this tool, you can perform sequencing! HOW? Establish a unique signal for each of the 4 nucleosides (add fluorescent tag to each one!) ddATP ddGTP ddTTP ddCTP Now that you have a collecIon of labeled ddNTPs… (normal) (terminator) Add them into DNA polymerase reacIons at low concentraIon (they will get incorporated about 1% of the Ime). They will terminate the reacIons! But, they will terminate the reacIons at different Imes ­ this is the key! There will be different lengths of DNA generated that are now different lengths. IMPORTANT: you start with single stranded template DNA Then, you resynthesize the complementary strand with a unique end (terminator) You need: template, primer, polymerase, ddNTPs, and a means to detect them! Mixing all four labeled ddNTPs together in a reacIon will let you know the complete sequence Now that you have thousands of terminated strands of DNA, what do you do? Old school answer: run a gel! New school answer: run pieces through a capillary tube ajached to a fluorescence reader. SIll based on size! As each one passes through the detector, the excitaIon is measured. Called the Sanger method, aqer Frederick Sanger, its inventor. He was awarded his second Nobel prize in 1980 for this discovery! Automated sequencing: fast and inexpensive (relaIvely) Can perform about 24 runs per day (gexng faster all the Ime) and sequence an organism’s genome for a few thousand dollars, although this obviously depends on the size of the genome (yeast: 12 million bps, zebrafish: 1.7 billion bps, humans: 3 billion bps)! More on sequencing Old school: New school: Everything is basically automated now ­ faster, cheaper, more efficient, and less labor ­intensive. What used to take a very long Ime (data processing) is now done in seconds by computers. Sequencing: the animaIon What if you have a piece of DNA that is too big for one sequencing reacIon? primers Regions sequenced Design new primers! Regions sequenced Known as “walking”: successively building sequence on the basis of new informaIon Strategies to sequence the whole genome Need: A collecIon of cloned DNA fragments whose sequences overlap Shotgun sequencing: Done in a similar way as on last slide! Recombinant DNA •  Overview and geneIc concepts •  Cloning –  Tools •  RestricIon endonucleases •  Ligase •  Polylnkers (MCS) –  Building a clone •  Libraries –  A source of clones –  Screening •  •  •  •  Defining what you’ve cloned PCR Learning from hybridizaIon: blots, in situs, etc Expression cloning Polymerase chain reacIon (PCR) •  Uses: cloning, forensics, idenIficaIon •  SelecIve amplificaIon of DNA sequences •  Relies on knowledge of DNA sequence (and in vitro DNA denaturaIon/hybridizaIon + polymerizaIon) Invented by Kary Mullis (and colleagues) at Cetus corporaIon (rumored to have been thought of while he was under the influence of certain psychotropic chemicals... Many patent bajles have gone on ­ the pharmaceuIcal giant Roche currently owns all patents on PCR! PCR: what you need •  Template (target DNA) •  SyntheIc oligonucleoIde primers (with homology to the ends of the targeted sequence) •  dNTPs •  Thermostable DNA polymerase (Taq, from Thermus aqua4cus, a bacteria that lives in hot springs) •  Means of controlling the reacIon (thermocycler) PCR: nuts and bolts Concept: Denature template DNA (with heat) with primers and polymerase present. Lower temperature (allow primers, which are in great excess) to anneal (base pair) to the DNA template. Elongate the primers (make DNA copy) Every cycle is a doubling of DNA. Net result? ExponenIal increase in transcript numbers (about 1 million fold in 20 cycles)! PCR: nuts and bolts Concept: Denature template DNA (with heat) with primers and polymerase present. Lower temperature (allow primers, which are in great excess) to anneal (base pair) to the DNA template. Elongate the primers (make DNA copy) Every cycle is a doubling of DNA. Net result? ExponenIal increase in transcript numbers (about 1 million fold in 20 cycles)! PCR uses •  •  •  •  Forensics DiagnosIcs ­ classic experiment! Cloning ­ check out figure 5 ­24 Probe preparaIon –  Just incorporate a labeled dNTP into the reacIon (radioacIve or fluorescent) •  LimitaIons –  Fidelity ­ has a higher rate of errors than in vivo (although some polymerases do have proofreading capability) –  Size ­ can’t get huge genes PCRed in one reacIon (varies by polymerase). SomeImes need to “walk” to generate a long clone. PCR ­ the animaIon Important ­ this will probably answer some of your conceptual quesIons about PCR! Somehow, it’s always faster on CSI… (and, the labs look way cooler) hjp://www.youtube.com/watch?v=6iFDphWXjw4 Working with clones and sequences •  Southern blot –  Detects DNA fragments in genomic samples •  Northern blot –  Detects specific RNA fragments •  In situ (in posiIon) hybridizaIon –  Detects DNA or RNA in specific locaIons •  Microarrays –  Gene expression ...
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This note was uploaded on 10/12/2011 for the course BIMM 100 taught by Professor Pasquinelli during the Summer '06 term at UCSD.

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