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Biotechnological tools & techniques

Biotechnological tools & techniques. Mrs. Ciampini. Molecular Biologists. Use tools!! Biological organisms Biological molecules To investigate genetic disorders, Alter the genetic make up of organisms Analyze DNA evidence in criminal investigation. Alter genes & respective proteins .

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Biotechnological tools & techniques

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  1. Biotechnological tools & techniques Mrs. Ciampini

  2. Molecular Biologists Use tools!! • Biological organisms • Biological molecules • To investigate genetic disorders, • Alter the genetic make up of organisms • Analyze DNA evidence in criminal investigation. • Alter genes & respective proteins

  3. Recombinant DNA Fragment of DNA composed of sequences originating from two+different sources.

  4. Restriction endonucleases Technique #1

  5. Restriction endonucleases (A.K.A. restriction enzymes) What are they? Molecular scissors What they do? • Cut double stranded DNA at specific sites. Why are they useful? • Can cut DNA in precise and predictable ways.

  6. RECOGNITION SITES • Each restriction enzyme recognizes a specific sequence of nucleotides (“RECOGNITION SITE”). • 4-8 base pairs long • Complementary palindrome sequence

  7. PALINDROME?? • Read the same forward as backwards. • LEVEL, RACECAR, HANNAH... • “GO HANG A SALAMI IM A LASAGNA HOG!” 5’- GAATTC -3’ 3’ -CTTAAG -5’ Key: Both read the same 5’3’

  8. HOW DO THEY WORK? scan: DNA molecule stop: recognition site bind: recognition site disrupt: phosphodiester bonds (via hydrolysis reaction) which may lead to disruption in hydrogen bonds too! result: • cut within DNA strand • two DNA fragments (instead of one)

  9. LIST OF RESTRICTION ENZYMES AND THEIR RECOGNITION SITES

  10. BLUNT VS. STICKY Different restriction enzymes produce different ends of DNA fragments. Key: Look at the ends! Sticky:short single stranded overhang Blunt:fully based paired

  11. STICKY ENDS MORE USEFUL TO MOLECULAR BIOLOGISTS!

  12. BIOLOGICAL MOLECULES! • Restriction enzymes – isolated and purified solely from bacteria • “crude immune system”

  13. NAMING Restriction enzymes are named based on the bacteria in which they are isolated. For example, EcoR1: E Escherichia (genus)co coli (species)RRY13 (strain)1       First identified (Order ID'd in bacterium)

  14. HAMILTON SMITH, 1970 • Accidental discovery of these site-specific restriction enzymes!! • How do some bacteria resist invasion of viruses? • Saw: Virus bacteria degrades, host does not – why? • Hypothesis: enzyme responsible? • Isolated: Hind11 • Nobel Prize, 1978 • More than 2500 restriction enzymes found!! • 200 target sites!

  15. METHAYLASES • Need to distinguish between foreign DNA & own DNA! • Why? How do they do this? • Methylases – specific enzymes found in P&E • Modify recognition sites • Add methyl group (CH3) on one bases to OWN DNA. • ROAD BLOCK – no cut Foreign DNA is NOT methylated! NO DEFENSE!!

  16. DNA LIGASES • DNA LIGASES - JOIN DNA!! • TWO different fragments created with SAME restriction enzyme = natural attraction!! • Why?

  17. DNA LIGASES Hydrogen Bonds FORM between complementary strands. (Engagement) Not Stable Enough... PHOSPHODIESTER LINKAGE BETWEEN BACKBONES OF DOUBLE STRANDS NEEDED... (Marriage) DNA LIGASE TO THE RESCUE!! (Priest) Condensation Reaction – DNA ligase drives out a molecule of water and reforms phosphodiester linkage.

  18. WE CAN USE RESTRICTION ENZYMES TO EXCISE A DESIRED GENE FROM A STRAND OF DNA!

  19. Once desired gene has been excised from source DNA... it must be separated from remaining unwanted fragments!

  20. GEL ELECTROPHORESIS TECHNIQUE #2

  21. WHAT IS IT? • Technique to separate charged molecules on the basis of size. • Sorting through a gel meshwork.

  22. MOLECULAR SEIVE Restriction Enzymes – cut DNA into fragments of different length. Migration: Shorter fragment – faster travellers Longer fragments – slower travellers Analogy: Forest Runs Large Group vs. Pair

  23. GEL: • Rectangular slab • Agrose (seaweed) • Polyacrylamide (artificial) • WELL: depression at one of the gel. • SOLUTION: • Different-sized fragments of DNA + loading dye (visualization).

  24. DIRECT CURRENT APPLIED: Negative charge at end of wells Positive charge at opposite end. DNA = negative (each nucleotide possess a phosphate group that carries a -1 net charge) IF THIS IS SO, IN WHICH DIRECTION WILL DNA MIGRATE?

  25. ETHIDIUM BROMIDE • carcinogenic • flat molecule • inserts itself among the rungs of the ladder of DNA • fluoresces under UV light. • Fluoresces: • glows under UV light • excitation of electrons. HOW DO WE KNOW HOW LONG THESE FRAGMENTS ARE?

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