Presentation on theme: "Manipulating DNA: tools and techniques"— Presentation transcript:
1Manipulating DNA: tools and techniques Chapter 12Key Knowledge:tools and techniques: gel electrophoresis; DNA profiling; DNA sequencing; DNA recombination; DNA amplification; gene cloning, gene transformation; gene delivery systems;
2Mitochondrial 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
4Mitochondrial DNA (mtDNA) see Pages 419 – 421 of textbook “Murders at Ekaterinberg”.
5Tools for Genetic Engineering Action‘Tool’Cut 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)
6Restriction EnzymesThe molecular biology revolution started with the discovery of restriction enzymes (restriction endonucleases)Enzymes cleave DNA at specific sitesThese 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)
7Restriction Enzymes Restriction enzymes cut at specific sites Some form ‘blunt ends’ (straight cut) others form ‘sticky ends’ (staggered cut). e.g. EcoRI
9Gel Electrophoresis A technique used to separate DNA fragments by size The 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.
10Dye added to the DNAMakes the sample visible when it is put into the agarose wells
11Buffer solution added to the tank This ensures that the electric current goes through the whole tank and that maintains that ions can move in the solution
12DNA samples loaded into wells Glycerol also in the loading dye
13Electrical current applied to the chamber Safety 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 electrodeOnce the dye has moved through the gel to the buffer, the electrical current is switched off and gel is removed from the tray
14DNA is stained using ethidium bromide Gel 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 wells
15ProbesA 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).
19Transporting DNA into cells DNA 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.
20Steps to Transporting DNA into a Target Cell DNA 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.
27Gene 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
29Gene Cloning Making multiple copies of a gene The 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.
30Polymerase 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.
34Gene Transformation First identified by Griffith in 1928 Defined as the take up of naked DNA by cellsOccurs naturally in bacteria, yeast and some plantsMay be inducedMix bacteria with CaCl2 solutionPlace in ice (0°C)Then place at 37°CCells are now competent
37Gene Delivery SystemsGene Delivery is a process of inserting foreign DNA into host cellsThere are many different processesViralNon-viralIt is the key to Gene Therapy
38Gene Delivery Systems – Viral Viral 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 cell
39Gene Delivery Systems – Non Viral Plasmidsmicroinjectiongene gunimpalefectionhydrostatic pressureelectroporationcontinuous infusionsonicationlipofection