Slide 1Slide 2Slide 3Slide 4Slide 5Slide 6Slide 7Slide 8Slide 9Slide 10Slide 11Slide 12Slide 13Slide 14Slide 15Slide 16Slide 17Slide 18Slide 19Slide 20Slide 21Slide 22Slide 23Slide 24Slide 25Slide 26Slide 27Slide 28Slide 29Slide 30Slide 31Slide 32Slide 33Slide 34Slide 35Slide 36Slide 37Slide 38Slide 39Slide 40Slide 41Slide 42Slide 43Slide 44Slide 45Slide 46Slide 47Slide 48Today: BiotechnologyOver 600 recent transposon insertions were identified by examining DNA from 36 genetically diverse humans.Tbl 1 Which transposable elements are active in the human genome? (2007) Ryan E. Mills et al. Trends in Genetics 23: 183-191DNA fingerprinting using RFLPsVisualizing differences in DNA sequence by using restriction enzymesSequence 1Sequence 2Restriction Enzymes cut DNA at specific sequencesFig 18.1EnzymeRecognitionSequence CutEcoRI 5'GAATT C3'CTTAAG5'---G AATT C---3'3'---CTTAA G--- 5'BamHI 5'GGAT CC3'CCTAGG5'---G GAT CC---3'3'---CCTAG G---5'HindIII 5'AAGCTT3'TTCGAA5'---A AG CTT---3'3'---TTCGA A--- 5'TaqI 5'TCGA3'AGCT5'---T CGA---3'3'---AGC T---5'AluI 5'AGCT3'TCGA5'---AG CT---3'3'---TC GA---5'Examples of some restriction enzymes…tbl 18.3Visualizing differences in DNA sequence by using restriction enzymesSequence 1Sequence 2Fig 20.5+.6Separating DNA on a gel by sizeFig 20.6•Gel electrophoresisFig 24.21The different sized bands can arise from different cut sites and/or different number of nucleotides between the cut sites.Fig 22.23Sequence 1Sequence 1Sequence 2Sequence 2DNA fingerprintingDNA fingerprintingDNA fingerprintingCan DNA be obtained from hair?How can DNA be obtained from such a small sample?The inventor of PCRPolymerase Chain Reaction:amplifying DNAFig 18.6Polymerase Chain ReactionFig 18.6Polymerase Chain Reaction:Primers allow specific regions to be amplified.Fig 18.6The inventor of PCRPCR animation http://www.dnalc.org/ddnalc/resources/pcr.htmlAreas of DNA from very small samples can be amplified by PCR, and then cut with restriction enzymes for RFLP analysis.Genetic Engineering: Direct manipulation of DNAFig 18.2Bacteria can be modified or serve as intermediatesFig 18.2a typical bacteriaBacterial DNAplasmid DNAA typical bacterial plasmid used for genetic engineeringtbl 18.2Moving a gene into bacteria via a plasmidFig 18.2Bacterial DNAplasmid DNAWhat problems exist for expressing eukaryotic gene in bacteria?Reverse transcriptase can be used to obtain coding regions without introns.Fig 18.4After RT, PCR will amplify the gene or DNAFig 18.6Moving a gene into bacteria via a plasmidRT and PCRFig 18.2Restriction Enzymes cut DNA at specific sequencesFig 18.1Restriction enzymes cut DNA at a specific sequenceFig 18.1Cutting the plasmid and insert with the same restriction enzyme makes matching sticky endsFig 18.1A typical bacterial plasmid used for genetic engineeringUsing sticky ends to add DNA to a bacterial plasmidFig 18.1Transformation of bacteria can happen via several different methods.tbl 6.1Bacteria can take up DNA from the environmentFig 9.2Tbl 6.1Transformation of bacteria can happen via several different methods all involving perturbing the bacterial membrane:•Electroporation•Heat shock•Osmotic StressHow can you know which bacteria have been transformed, and whether they have the insert?Fig 18.1Resistance genes allow bacteria with the plasmid to be selected.Bacteria with the resistance gene will survive when grown in the presence of antibioticFig 20.5Is the insert present?Plasmids with the MCS in the lacZ gene can be used for blue/white screening…Fig 18.1A typical bacterial plasmid used for genetic engineeringIntact lacZ makes a blue color when expressed and provided X-galactoseWhen the lacZ gene is disrupted, the bacteria appear whiteBlue/white screening:Transformed bacteria plated on antibiotic and X-gal plates.Each colony represents millions of clones of one transformed cell.Fig 18.1Successful transformation will grow a colony of genetically modified bacteriaFig 18.1Inserting a gene into a bacterial plasmidRT and/or PCRFig 18.1Millions of HectaresTexas =70 haBacteria can be used to transform plantsGlobal area planted with GM