3 BackgroundProject will have you cloning the gene that codes for the enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH)GAPDH is a housekeeping gene necessary for survivalGAPDH is an enzyme that is crucial for glycolysis to occur
5 GAPDH can be easily isolated in cells Is made up of four subunits that are either identical (homotetramer) or in pairs of slightly different proteins (heterodimer)Has two domains: amino terminal region binds to NAD+ while the carboxy terminal region has the dehydrogenase activityDoes two things:Removes H+ from GAP and transfersit to NAD+Adds second Phosphate to GAP
6 GAPDH genesFound in the cytosol (glycolysis) and in the chloroplast as part of photosynthesisIsozymes coded for on nuclear DNAGAPC denotes the gene that codes for cytosolic GAPDH and is the gene that we will study.The GAPC protein is a heterodimer.
8 Big picture for this unit Isolate GAPC gene from plantsAmplify the GAPC gene by nested PCRAssess the results of the PCRPurify the PCR product containing GAPCLigate (insert) GAPC gene into plasmid vectorTransform bacteria with new plasmidIsolate plasmid from bacteriaConfirm plasmid by restriction digestsPrepare plasmid DNA to be sequenced by outside facilityAnalyze sequence of your GAPC gene using bioinformatics
9 Nucleic Acid Extraction Task is to separate DNA from rest of the cellular components, including membranes, proteins, and enzymesMust also remain in tact after extractionPlant cells also have a cell wall to disruptNucleases can digest DNAAcidic contents of organelles can damage DNASome plants have polyphenols that bind to DNA rendering it useless for experiments
10 Basic Steps of DNA Extraction Harvest cells from fresh, young plantsGrind cells to physically disrupt tissue & cell wallsLyse cells to disrupt membranesRemove cellular debris by centrifugationDigest remaining cellular proteins
11 Basic Steps of DNA Extraction Purify DNA by ion-exchange chromatography to remove contaminantsConcentrate DNA by ethanol precipitationDetermine purity and concentration of DNA with UV Spec
12 Lysis Buffers EDTA to destabilize the membrane and inhibit nucleases Buffers to maintain pH since acids are released by organellesDetergent to dissolve membraneDTT denatures proteins
13 Polymerase Chain Reaction Rapidly creates multiple copies of a segment of DNAUses repeated cycles of DNA synthesis in vitroUsed in DNA fingerprinting, kinship analysis, genetic testing for mutations, and infectious disease for diagnosis
15 PCR players DNA template – targeted piece of DNA Primers – small segments of DNA that bind complementary upstream and downstream of the target on the templateTaq DNA polymerase – isolated from the Thermus aquaticus bacteria found in hotsprings of Yellowstone ParkDNA nucleotides in the form of deoxynucleoside triphosphates (dNTPs)Reaction Buffer – maintains pH for enzymes
16 General PCR ProcessDenaturation – split apart the two DNA strands by heating them to 95oC for 1 minAnnealing – primers bind to target sequence by cooling reaction to 40-60oC for 1 minExtension – Taq Polymerase extends the primers and copies each DNA template strand by heating to 72oC for 1 min
17 PrimersRequired for both sides of the target sequence (forward & reverse primer)Length of primer is generally nucleotidesG/C content and intra-complementarity are a concern when designing primersActually not a single primer for each but a mixture of primers (oligoprimers) if the sequence of the target is not knownIf amino acid sequence of gene product is used then degenerate primers must be usedInitial forward primer isGABTATGTTGTTGARTCTTCWGGB=G/T/C R=G/A (purines) W =A/T
18 Nested PCRInitial PCR primers are degenerate and based on a consensus sequenceThe chances that the initial primers will bind to sequences other than the target are highA second set of primers designed to be more specific to GAPC is usedThey are nested within the initial primers and are not degenerate thus much more specific to the GAPC gene
22 PCR purificationSmall impurities can have a negative effect on the ligation of the PCR product to vector DNAImpurities include unincorporated dNTPs, polymerases, primers and small primer-dimers.A PCR Kleen spin column will remove the impurities in less than 4 min.
23 Gene CloningCloning is the production of exact copies of a piece of DNA.It requires ligating (splicing) the PCR product into a cloning vector – often a plasmid DNAThe recombinant DNA of the ligation product can now be put into a cell to propagate (replicated)
24 Plasmids are good vectors: small (2,000 – 10,000 bp)circular, self-replicatinghigh copy numbermultiple cloning sites (MCS)selectable markers (Amp-resistance)screening (reporter genes, positive select)control mechanisms (lac operon)can handle the size of the insert
25 pJet1.3 blunted vector Designed for blunt-end cloning High copy number Contains Amp-resistant geneContains eco47IR gene which allows for positive selectionIt is 2,974 bp long
26 InsertsSticky ends have single strands of nucleotides on ends and are good for directional insertingBlunt ends have no singlestrands and thus are easierto insert but are nondirectional.
27 LigationT4 DNA Ligase catalyzes formation of phosphodiesterase bond between 3’ hydroxy on one piece and the 5’ phosphate on another piece.Requires ATP and Mg+2Insert to vector DNA ratio should be 1:1Proofing reading DNA polymerase removes dangling 3’A of PCR product
28 Products of Ligation Self-ligation of vector Ligation of vector to primer-dimersLigation of multiple insertsSelf-ligation of insertsLigation of one insert into vector
29 TransformationOnce PCR product (insert) has been ligated into a plasmid, the plasmid be introduced into a living bacterial cell to replicate.Two methods of transformation:ElectroporationHeat ShockBoth methods make cells competent - able to take up plasmids
30 Transformation Steps Wash away growth media from cells Place cells in ice cold calcium chloride which most likely hardens the cell membraneAdd plasmid to cellsMove cells to hot environment (usually 42oC) causes membrane pores to open so plasmid can enterAdd nutrient media to cells to allow them to recover from stressPlate cells on selective growth plates (Amp and IPTG (increases expression of ampr gene)
31 Microbial CulturingPick a colony from the transformed cells to innoculate a liquid cultureLiquid culture (broth) must have selective antibiotic (Amp) in it.Choose a single colony from the plateUnder favorable conditions, a single bacteria divides every 20 minutes and will multiply into billions in 24 hours
32 Plasmid PurificationTo confirm that the engineered cells have been transformed with the correct DNADifferent methodsLysozyme MethodAlkaline Cell Lysis MethodColumn Methods (Aurum)
33 Plasmid prepsSpectrophotometer determination of culture density. Take OD600 of culture (equal to about 8x108 cells/mlAurum column can process up to 12 OD●ml of bacterial host cellsCells disrupted with a lysis bufferDNA binds to membrane of column, is washed and then eluted with aqueous buffer.
34 Restriction Digests DNA cut with restriction enzymes Evolved by bacteria to protect against viral DNA infectionEndonucleases -cleave within DNA strandsOver known enzymes
35 Restriction DigestsEach enzyme cuts DNA at a specific sequence= restriction siteMany of the restriction sites are 4 or 6-base palindrome sequencesEnzyme cutsFragment 2Fragment 1
37 Restriction Digest Restriction Buffer provides optimal conditions: NaCl provides correct ionic strengthTris-HCl provides proper pHMg+2 is an enzyme co-factorBody temperature (37oC) is optimalToo hot kills enzymeToo cool takes longer digestion timeOdy
38 DNA SequencingDetermining the exact order of the nucleotide sequence in a DNA molecule.Use to take days, now takes hoursHave sequences of entire genones for over 700 organisms
39 Sanger Method Prepare single-stranded DNA template to be sequenced Divide DNA into four test tubesAdd primer to each tube to start DNA synthesisAdd DNA polymeraseAdd labeled deoxynucleotides (dNTP) in excess. Labeled with radioactive or fluorescent tagsAdd a single type of dideoxynucleotides (ddNTPs) to each tube. When incorporated in sythesized strand, synthesis terminates.Allow DNA synthesis to proceed in each tubeRun newly synthesized DNA on a polyacrylamide gel
40 Reading the SequenceIn the tube with the ddTTP, every time it is time to add a T to the new strand, some Ts will be dTTP and some will be ddTTP.When the ddTTP is added, then extension stops and you have a DNA fragment of a particular length.The T tube will, therefore, have a series of DNA fragments that each terminate with a ddTTP.Thus the T tube will show you everywhere there is a T on the gelSame thing happens in all tubesRead gel from top to bottom looking at all four lanes to get the sequence.
41 Automated SequencingDye-terminator sequencing labels each of the ddNTPs with a different color fluorescent dye.Now reaction can be run in one tubeUse capillary electrophoresis rather than the standard polyacrylamide slab gel.When DNA fragment exits gel, the dyes are excited by a laser and emit a light that can be detected .Produces a graph called a chromatogram or electopherogram
43 BioinformaticsComputerized databases to store, organize, and index the data and for specialized tools to view and analyze biological dataUses includeEvolutionary biologyProtein modelingGenome mappingDatabases are accessible to the publicAllow us to record, compare, or identify a DNA sequence