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Biotechnology

Biotechnology. DNA technology. DNA diagnostics DNA therapy. DNA isolation. Cell lysis Removal of proteins DNA precipitation by ethanol DNA dilution in water or buffer. DNA diagnostics. – PCR (amplifying part of DNA up to 10 6 copies)

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Biotechnology

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  1. Biotechnology

  2. DNA technology • DNA diagnostics • DNA therapy

  3. DNA isolation • Cell lysis • Removal of proteins • DNA precipitation by ethanol • DNA dilution in water or buffer

  4. DNA diagnostics – PCR (amplifying part of DNA up to 106 copies) – sequencing, alignment of all nucleotides – restriction digestion, testing specific nucleotides – reverse transcription – cDNA – qPCR – blotting – Southern (DNA) Northern (RNA) Western (protein)

  5. PCR Polymerase Chain Reaction

  6. 1. 2. 3.

  7. PCR cycle

  8. PCR cycling (30 cycles) Cycle: 1. n=21 2. n=22 n = number of gene copies (exponential growing) 3. n=23

  9. DNA Visualization and Separation Gel sieve structure of polymer molecules with pores Fluorescence dye binding to DNA and excites photons under UV-exposure

  10. DNA Visualization and Separation Gel Electrophoresis • the movement of charged molecules in electric field • the movement direction from – to + • DNA-rate in gel depends on DNA-fragment lengthin indirect proportion result under UV-light

  11. Sequencing Genome sequencing lines in vertical sequence gel:

  12. Sequencing

  13. Restriction digestion Bacterial restriction enzymes recognize palindromic sequence in DNA.

  14. Restriction digestion Detection of mutation • mutative DNA – digestion (two small fragments) • normal DNA – non- digestion (one large fragment) results after gel electrophoresis read under UV-light

  15. Complementary DNA (cDNA)of eukaryotic organisms • in laboratory, it results from reverse transcription • in certain gene: cDNA < DNA • It is quantified by qPCR - marker of gene expression

  16. CELL DIFFERENTIATION: • arises because cells make and accumulate different sets of RNA and protein molecules • that is, they express different genes DNA of all cells in the body of one individual is identical ! ! !

  17. Blotting: transfer a substance from gel to membrane • Mechanisms: • Capillary attraction • electrophoretic transfer

  18. Blotting:  a basic molecular biology technique originally created by Edwin Southern (1975)        for locating gene specific sequences on DNA fragments. SOUTHERN BLOTTING: is used to locate and identify genes on DNA. DNA restriction fragments are electrophoretically separated. The fragments are blotted onto membranes, where the DNA bonds. Hybridization with labeled DNA probes & localizing target DNAs. NORTHERN BLOTTING: a variation on Southern blotting. RNAs are separated by electrophoresis, transferred to membranes, and probed with a labeled DNA probes. WESTERN BLOTTING: another variation on Southern blotting. Proteins are separated by electrophoresis, transferred to membranes, and probed with an antibody that binds to the protein you are interested in locating.

  19. DNA therapy Bacteria or yeast produce human proteins coding by human genes: - coagulation factor VIII - insulin - growth hormone

  20. DNA cloning

  21. Cloning organisms • Reproductive • Therapeutic

  22. Literature Biology, eighth edition, Campbell, Reece Unit three: Genetics Chapter 20: Biotechnology Pages 396 – 425

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