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PCR-technique Applications

PCR-technique Applications. by E. Börje Lindström. This learning object has been funded by the European Commissions FP6 BioMinE project. DNA synthesis (replication). General aspects (in vivo):. semi conservative. - 1 old + 1 new strand. template. - the strand that is copied.

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PCR-technique Applications

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  1. PCR-techniqueApplications by E. Börje Lindström This learning object has been funded by the European Commissions FP6 BioMinE project

  2. DNA synthesis (replication) General aspects (in vivo): • semi conservative - 1 old + 1 new strand • template - the strand that is copied • replication • Synthesis in direction 5’3’ • the new strand has a free OH-group • DNA polymerase III • the enzyme needs a primer with a free OH-group to start • the primer is RNA (in vivo) or DNA (in vitro)

  3. PCR • Pre-requisite: • a sequence of nucleotides must be known on both the strands • primers (15-25 oligonucleotides) can be made • The process: 1) Heat denature the DNA molecule:  ssDNA 2) Cool and add: - primers, polymerase and dNTP • binding of primers to ssDNA (annealing) • synthesis of DNA from the primers • incubate a certain time - exponentially increase of the DNA-molecules 3) Repeat the process: 

  4. PCR, cont.

  5. PCR, cont.

  6. PCR, cont.

  7. PCR, cont. • Note! 1) Taq-polymerase (Thermus aquaticus) is thermo stabile  the reaction can be run at 70-72C. 2) The product increases exponentially- the template linearly 3) Problems: • contamination by ’foreign’ DNA-molecules • mistakes in DNA synthesis  use a polymerase with proof-reading • use separate pipettes for PCR

  8. Some application of PCR 1) Medicine: • diagnose of pathogenic micro organisms • diagnose of early stages of a bacterial infection • trace viruses • trace chromosomal aberrations 2) Juridical/forensic medicine: • blood • saliva • fatherhood If positive PCR Found guilty!

  9. Some application of PCR, cont. 3) Molecular biology: • DNA sequencing possible after PCR - low amount of DNA  high amount • Determine chromosomal aberrations • in vitro mutagenesis • construction of vectors ( + restriction sites) 4) Microbial ecology

  10. Microbial ecology What is studied? • Bio-diversity - species variation • Microbial activity • nitrogen fixation • sulphate reduction etc.

  11. Bio-diversity Cultivable micro-organisms: - < 1% of natural populations • Enrichment - uses selection and counter selection • the choice of starting material important • selection of growth medium (amount and chemical form of the nutrients) • selection of environment (temp., pH etc.) • Isolation • single-cell-colonies on plates • casting in melted agar (tubes) • most-probable-number (MNP) • use laser tweezers Note! Important to confirm the purity!

  12. Bio-diversity, cont. Non-cultivable micro-organisms: • Quantifying: • total amount of m.o. • specific groups of m.o. • specific metabolic processes Staining techniques: • DAPI (4’,6-diamido-2-phenylindole) • fluorescence • binds to nucleic acids • all types of cells (alive and dead) • total amount of cells

  13. Bio-diversity, cont. • Live/Dead BacLightTM: • (contains propidium iodide) • green cells  cells alive • red cells  dead cells • Fluorescent antibodies: • identifying specific m.o. • e.g. clinically, pathogenic m.o. Note! All staining techniques use microscopy  no information about the genetically variation in the population.  Molecular methods needed!

  14. Bio-diversity, cont. Molecular methods: FISH (fluorescent in situ hybridization) • Species composition of a sample: • Use of: • group specific sequences in 16S rRNA as probes (species, ….domains) • different fluorescent dyes attached to the probe • the cells are fixated and made permeable to the probe/s • hybridization direct to the ribosomes  The whole cell appear fluorescent

  15. Bio-diversity, cont. • Identification of specific genes: • Use of: • a fluorescent probe against part of a gene • treat the cells as previously for FISH  The gene is present in the population if positive staining!

  16. Bio-diversity, cont. • Identification of expressed genes: • Use of: • ISRT (in situ reverse transcriptase + FISH • Probe 1 against a specific mRNA molecule • Binding + reverse transcription  complementary DNA-strand produced • DNA synthesis with PCR • a fluorescent dye is added to get probe 2 • FISH

  17. Bio-diversity, cont. • Phylogeny studies: • extraction of total DNA in the sample • amplify by using PCR • group specific primers (16S rRNA genes) How to separate? • Use: • DGGE (denaturing gradient gel electrophoresis) • resolving genes of the same size but differing in sequences

  18. Bio-diversity, cont. • Based on: • a denaturing substance/ agent • heat or • urea/form amid mixture  ds-DNA  ssDNA at a special conc..  Each band can be isolated and sequenced!

  19. DNA sequencing Two methods have been developed: • Maxim and Gilbert method • Sanger dideoxy method • 4 reaction mixtures (tubes) are used • nowadays only one tube! 20 Sanger: • ssDNA(/RNA) • primers • DNA-polymerase • a mixture of dATP, dCTP, dGTP, and dTTP • Tube 1: + ddATP • Tube 2: + ddCTP • Tube 3: + ddGTP • Tube 4: + ddTTP

  20. DNA sequencing, cont.  Electrophoresis  Analysis Read from bottom: C, A, T, G, C, C, A

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