1 / 35

DNA Technology and the Human Genome

DNA Technology and the Human Genome. Chapter 12. Bacteria as Tools for Manipulating DNA. DNA Technology. The Human Genome Project Better understand diseases and their causes Gene therapy The production of vaccines, cancer drugs, and pesticides

emmet
Télécharger la présentation

DNA Technology and the Human Genome

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  1. DNA Technology and the Human Genome Chapter 12

  2. Bacteria as Tools for Manipulating DNA

  3. DNA Technology • The Human Genome Project • Better understand diseases and their causes • Gene therapy • The production of vaccines, cancer drugs, and pesticides • Engineered bacteria that can clean up toxic wastes • Restore damaged ecosystems

  4. DNA enterscell Fragment ofDNA from anotherbacterial cell Bacterial chromosome(DNA) Bacteria as a Tool for Manipulating DNA • In nature, bacteria can transfer DNA in three ways • Transformation, the taking up of DNA from the fluid surrounding the cell • Can even take up DNA from dead cells

  5. Conjugation, the union of cells and the DNA transfer between them Transduction, the transfer of bacterial genes by a phage Mating bridge Phage Fragment ofDNA from anotherbacterial cell(former phagehost) Donor cell(“male”) Recipient cell(“female”) Figure 12.1B Bacteria as a Tool for Manipulating DNA

  6. Donated DNA Degraded DNA Crossovers Recipient cell’schromosome Recombinantchromosome • The transferred DNA is then integrated into the recipient cell’s chromosome

  7. Bacterial plasmids can serve as carriers for gene transfer An F factor is a DNA segment in bacteria that enables conjugation and contains an origin of replication The F factor starts replication and transfers part of the chromosome F factor (integrated) Male (donor) cell Origin of F replication Bacterial chromosome F factor startsreplication andtransfer of chromosome Recipient cell Only part of thechromosome transfers Recombination can occur Bacterial Phages as Carriers

  8. An F factor can exist as a plasmid, a small circular DNA molecule separate from the bacterial chromosome R plasmids carry genes for resistance of antibiotics and that is how bacteria can become resistant F factor (plasmid) Male (donor) cell Bacterial chromosome F factor startsreplication andtransfer Plasmids Plasmid completestransfer andcircularizes Cell now male

  9. Plasmids are used to customize bacteria • Plasmids are key tools for DNA technology • Researchers use plasmids to insert genes into bacteria • Plasmids are obtained from other bacteria • Desired DNA inserted into plasmid • Bacteria takes up DNA • Can be used for several applications

  10. Cell containing geneof interest 1 Bacterium Plasmidisolated 2 DNA isolated 3 Gene inserted into plasmid Bacterialchromosome Plasmid Gene ofinterest Recombinant DNA(plasmid) DNA 4 Plasmid put intobacterial cell Recombinantbacterium 5 Cell multiplies withgene of interest Copies of gene Copies of protein Gene for pestresistanceinserted intoplants Clones of cell Protein used to make snow format highertemperature Gene used to alter bacteriafor cleaning up toxic waste Protein used to dissolve bloodclots in heart attack therapy

  11. Restriction enzymerecognition sequence 1 DNA Restriction enzymecuts the DNA intofragments Restriction enzymecuts the DNA intofragments 2 Sticky end Addition of a DNAfragment fromanother source 3 Two (or more)fragments sticktogether bybase-pairing 4 DNA ligasepastes the strand 5 How they insert DNA into plasmids • Enzymes are used to “cut and paste” DNA • Restriction enzymes cut DNA at specific points • Recognize specific sequences • Make ‘sticky ends’ • DNA ligase “pastes” the DNA fragments together • Catalyst for hydrogen bonds • The result is recombinant DNA

  12. 1 Isolate DNAfrom two sources Human cell E. coli 2 Cut both DNAs with the same restriction enzyme Plasmid DNA Gene V Sticky ends 3 Mix the DNAs; they joinby base-pairing 4 Add DNA ligaseto bond the DNA covalently Recombinant DNAplasmid Gene V 5 Put plasmid into bacteriumby transformation 6 Clone the bacterium Cloning genes using recombinant plasmids • Bacteria take the recombinant plasmids and reproduce • This clones the plasmids and the genes they carry • Products of the gene can then be harvested • Make large amounts of a desired gene so that its protein can be manufactured

  13. Genome cut up with restriction enzyme Recombinantplasmid Recombinantphage DNA OR Phage clone Bacterialclone Phage library Plasmid library Genomic Libraries • Recombinant DNA technology allows the construction of genomic libraries • Genomic libraries are sets of DNA fragments containing all of an organism’s genes • Copies of DNA fragments can be stored in a cloned bacterial plasmid or phage

  14. Other Tools of DNA Technology

  15. CELL NUCLEUS Exon Intron Exon Intron Exon DNA ofeukaryoticgene Transcription 1 RNA transcript RNA splicing(removes introns) 2 mRNA Isolation of mRNAfrom cell and additionof reverse transcriptase;synthesis of DNA strand 3 TEST TUBE Reverse transcriptase Breakdown of RNA 4 cDNA strand Synthesis of secondDNA strand 5 cDNA of gene(no introns) Using mRNA as a Template • Researchers can use mRNA as a template to isolate the gene that makes that mRNA • Reverse transcriptase can be used to make smaller cDNA libraries • These contain only the genes that are transcribed by a particular type of cell

  16. Radioactiveprobe (DNA) Mix with single-stranded DNA fromvarious bacterial(or phage) clones Single-strandedDNA Nucleic Acid Probes • In order to find the bacteria or phage that contains the desired gene in a library • Radioactive probes of complimentary DNA sequences to the desired gene can be used to find the desired gene

  17. Bacterial colonies containingcloned segments of foreign DNA Radioactive DNA Transfercells tofilter 1 Solutioncontainingprobe Filterpaper Treat cellson filter toseparateDNA strands Add probeto filter ProbeDNA 2 3 Gene ofinterest Single-strandedDNA from cell Hydrogen-bonding Autoradiography 4 Colonies of livingcells containinggene of interest Developed film Compare autoradiographwith master plate 5 Master plate • Colonies are blotted on filter paper • Paper is treated to break up DNA • Probe is added • Paper laid on photographic film • Film is compared to colonies • Gene is identified

  18. Gel Electrophoresis • Physically sorts out macromolecules (DNA, RNA) on the basis of their charge and size • Current is run through the gel and since DNA is negatively charged it moves through the gel • The longer the DNA molecules are. The slower they move • Bands are made, each consisting of DNA molecules of one size

  19. Mixture of DNAmolecules ofdifferent sizes Longermolecules Powersource Gel Shortermolecules Glassplates Completed gel Gel Electrophoresis • Restriction fragments of DNA can be sorted by size

  20. Restriction Fragment Analysis • Everyone’s DNA sequence is different • Scientists can compare DNA sequences of different individuals based on the size of the fragments created by restriction enzymes • They can only use DNA that varies from person to person • When run on a gel it makes a distinct pattern

  21. 1 2 Allele 1 Allele 2 w Longer fragments Cut z x Shorter fragments Cut Cut y y DNA from chromosomes Restriction Fragment Analysis

  22. Restriction fragmentpreparation 1 Restrictionfragments Gel electrophoresis 2 Filter paper Blotting 3 Radioactive probe 4 Probe Detection of radioactivity(autoradiography) 5 Film Detecting Harmful Alleles • Radioactive single-stranded DNA complimentary strands are used to verify the presence of certain nucleic acid sequences known to code for harmful alleles

  23. PCR • The polymerase chain reaction (PCR) can quickly clone a small sample of DNA in a test tube • DNA sample mixed with DNA polymerase, nucleotides and other things and it replicates exponentially • Can replicate billions of clones within a few hours

  24. InitialDNAsegment 1 2 4 8 Number of DNA molecules PCR • Can copy a specific segment of DNA in a mass of DNA • Needs only minute amounts of DNA • Cannot produce large amounts of DNA • Scientists are using it to identify missing cases with bone marrow, prehistoric beasts etc

  25. The Challenge of the Human Genome

  26. The Human Genome • The 23 chromosomes in the haploid human genome contain about 3 billion nucleotide pairs • This DNA is believed to include about 35,000 genes and a huge amount of noncoding DNA (do not code for proteins) • About 97% of the total human genome • Much of the DNA between genes consists of repetitive DNA • Small sequences repeated over and over again on the same chromosome • Large sequences that are scattered throughout the genome

  27. The Human Genome Project • The Human Genome Project involves: • Genetic and physical mapping of chromosomes • Gene mapping and fragment analysis • DNA sequencing • Nucleotide sequence of the genes • Comparison of human genes with those of other species • Help the scientists interpret the human data

  28. Other Applications of DNA Technology

  29. Defendant’sblood Blood fromdefendant’sclothes Victim’sblood DNA Technology and The Criminal Court • Restriction fragment analysis using gel electrophoresis • Compare DNA from a crime scene to a sample from a suspect • Paternity

  30. Mass Production of Gene Products • Most are grown in cell culture using bacteria • E. coli can host the most plasmids, that is why it is used the most • Yeast is often better for manufacture of eukaryotic gene products • Study into using whole animals in gene product manufacture is underway

  31. Mass Production of Gene Products

  32. Agrobacteriumtumefaciens DNA containinggene for desired trait Plant cell 1 2 3 Tiplasmid RecombinantTi plasmid Insertion ofgene into plasmidusing restrictionenzyme and DNAligase Introductioninto plantcells inculture Regenerationof plant T DNA T DNA Plant with new trait Restriction site Genetically Modified Organisms (GMO’s) • New genetic varieties of animals and plants are being produced • A plant with a new trait can be created using the Ti plasmid

  33. GMO’s • “Golden rice” has been genetically modified to contain beta-carotene • This rice could help prevent vitamin A deficiency

  34. GMO’s and the Environment • Genetic engineering involves some risks • Possible ecological damage from pollen transfer between GM and wild crops • Pollen from a transgenic variety of corn that contains a pesticide may stunt or kill monarch caterpillars

More Related