Manipulating DNA: tools and techniques

Manipulating DNA: tools and techniquesChapter 12Key Knowledge:tools and techniques: gel electrophoresis; DNA profiling; DNA sequencing; DNA recombination; DNA amplification; gene cloning, gene transformation; gene delivery systems; Mitochondrial DNA (mtDNA)Inherited maternallyBehaves like prokaryotic DNAHaploidCircularMore than one copy in the mitochondriaUseful in family studiesmtDNA D-Loop extremely variable, accumulates many different mutations because it is not transcribed

Mitochondrial DNA (mtDNA)see Pages 419 421 of textbook Murders at Ekaterinberg.

Tools for Genetic EngineeringActionToolCut DNA into fragments at precise locationsRestriction Enzymes (pp. 422 423)Separate fragments by sizeElectrophoresis (pp. 423 424)Find particular DNA fragmentsProbes (pp. 424 425)Join DNA fragmentsLigase Enzyme (pp. 425 426)Transport DNA into cellsVectors (p. 426)Obtain multiple copies of geneGene Cloning (p. 428)Restriction EnzymesThe molecular biology revolution started with the discovery of restriction enzymes (restriction endonucleases)Enzymes cleave DNA at specific sites These enzymes are significant in two waysAllow a form of physical mapping that was previously impossibleAllow the creation of recombinant DNA molecules (from two different sources)

Restriction EnzymesRestriction enzymes cut at specific sitesSome form blunt ends (straight cut) others form sticky ends (staggered cut). e.g. EcoRI

Gel ElectrophoresisA technique used to separate DNA fragments by sizeThe gel (agarose or polyacrylamide) is subjected to an electrical fieldThe DNA, which is negatively-charged, migrates towards the positive poleThe larger the DNA fragment, the slower it will move through the gel matrixDNA is visualised using fluorescent dyes.Dye added to the DNA

10Makes the sample visible when it is put into the agarose wellsBuffer solution added to the tank

11This ensures that the electric current goes through the whole tank and that maintains that ions can move in the solutionDNA samples loaded into wells

12Glycerol also in the loading dyeElectrical current applied to the chamber

13Safety cover is put over the top and the current is switched onThe dye will migrate through the gel toward the positive electrode, as will the DNADepending on how much voltage is applied and how warm the gel is and size and shape of molecules will depend on how fast the mols move through the gelSmaller fragments will move easier so they will be closer to the positive electrode

Once the dye has moved through the gel to the buffer, the electrical current is switched off and gel is removed from the trayDNA is stained using ethidium bromide

14Gel is stained using ethidium bromide which binds to DNA it shows up as bands in UV lightDraw attention to the fact that small mols are at the bottom of the gel and large ones stay nearest to the wellsProbesA probe is used in order to find a specific sequence of DNA within a relatively large sample.Probes are usually labelled with a marker (e.g. radioactive, fluorescent, etc.) and are complementary to the target sequence.See Figure 12.9 in your textbook (pg 424).

LigaseThe enzyme Ligase is required to catalyse the joining of pieces of dsDNA at their sugar-phosphate backbone.

Transporting DNA into cellsDNA can be transported into cells through the use of vectors.Vectors are cellular agents that have the capacity to carry DNA and transport it into target cells.Bacteria are commonly used as vectors.Bacteria have a small circular piece of DNA called a plasmid.Steps to Transporting DNA into a Target CellDNA of the plasmid is cut using a restriction enzyme (the same restriction enzyme that the original DNA is cut with).The plasmid and the foreign DNA are mixed and their sticky ends pairDNA ligase makes the joins permanent.The plasmids that contain the recombinant DNA plasmid are then selected.

Steps to Transporting DNA into a Target Cell

Gene IsolationIt is easy to isolate the total DNA from human somatic cells46 chromosomes6 x 109 base pairs of DNA20,00 25,000 genesSequences of non-coding DNAGene IsolationHarder to isolate one gene or part of a gene

Gene IsolationLocate particular DNA fragments following separation by electrophoresis using a probe with a complementary base sequence

Gene IsolationSynthesis DNA from nucleotides: Use a DNA synthesiser to artificially manufacture a specific DNA sequence with lengths greater than 50 bases to be used as primers or probes

Gene IsolationMake a copy of DNA using a mRNA template: mRNA is isolated from specific cells and the enzyme Reverse Transcriptase is used to a make a complementary ssDNA strand. This is called copy DNA (cDNA). DNA Polymerase may be used later to convert the cDNA into dsDNA. This method only works for coding DNA: genes that produce mRNA

Gene CloningMaking multiple copies of a geneThe gene is copied and placed into a bacterial plasmidThe plasmid is inserted into the bacterial host cellInside bacterial host cell the plasmid and the gene make twenty copies of itselfThe bacterial cell copies itself every twenty minutes by binary fissionWithin several hours there are millions of copies of the bacterial host cell and the inserted gene.In some case the gene is switched on and the product (protein) is harvested.

Polymerase Chain Reaction (PCR)Polymerase chain reaction enables large amounts of DNA to be produced from very small samples.There is a repeating cycle of:Separation of double DNA strandsAnnealing of primers to the sequence to be amplified.Extension of DNA using the original DNA strand as a template.

The Reaction

THERMOCYCLERPCR tubeSeparation (heat to 95oC)Lower temperature to 56oC - Anneal with primersIncrease temperature to 72oC - DNA polymerase + dNTPs = extension of DNA

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Gene TransformationFirst identified by Griffith in 1928Defined as the take up of naked DNA by cellsOccurs naturally in bacteria, yeast and some plantsMay be inducedMix bacteria with CaCl2 solutionPlace in ice (0C)Then place at 37CCells are now competent

Gene Delivery SystemsGene Delivery is a process of inserting foreign DNA into host cellsThere are many different processesViralNon-viralIt is the key to Gene Therapy

Gene Delivery Systems ViralViral vectorsThey are good at targeting and entering cellsSome can be engineered to target specific types of cellsThey can be modified, so they do not replicate and destroy the cellGene Delivery Systems Non ViralPlasmidsmicroinjectiongene gunimpalefectionhydrostatic pressureelectroporation continuous infusionsonicationlipofection