Genetics in the News Recombinant DNA Technology

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Text of Genetics in the News Recombinant DNA Technology

  • 1. Genetics in the News

2. Recombinant DNA Technology

  • artificial manipulation of DNA.

3.

    • isolate DNA,
    • cut DNA into workable sized fragments,
    • amplify the fragments for storage and subsequent analysis,
    • identify and isolate specific sequences,
    • characterize by size genomic location and sequence.

Recombinant DNA Technology 4. Cutting the DNA

  • ~3,100,000,000 base pairs per human haploid genome,
    • average chromosome length is ~13 million base pairs,
  • DNA is cut with bacteria-derived enzymes,
  • The act of cutting is termed digestion .

5. Restriction Enzymes

  • proteins that recognize specific, short nucleotide sequences and cut DNA at those sites,
  • bacteria contain over 400 such enzymes and recognize and cut over 100 DNA sequences.

6. Bacteria Defense Mechanism

  • restriction enzymes are expressed in bacteria in order to protect against invasion by viruses,
  • bacterial protect their own DNA by chemically modifying recognition sites.

7. Palindromes

  • stir grits
  • no lemon, no melon

5 -------G-A-A-T-T-C--------3 3 -------C-T-T-A-A-G--------5 8. EcoRI 5 -------GA-A-T-T-C--------3 3 -------C-T-T-A-AG--------5 9. Sticky Ends

  • ...single-stranded DNA overhangs resulting from restriction digestion.

10. EcoRI 5 -------GA-A-T-T-C--------3 3 -------C-T-T-A-AG--------5 11. RsaI Rhodopseuomonas sphaeroides 5 -------G-TA-C--------3 3 -------C-AT-G--------5 12. 13. Restriction Fragment Length Sizes (predicted)

  • - IF -
  • 25% A25%T25% G25% C
  • and
  • Random Distribution of Nucleotides

- THEN - Distance between cut sites is equal to 4 nbases ,(n= number of base pairs in the recognition site) i.e.p(specific base) = .25 14. Average Restriction Fragment Length

    • n= 4,256 base pairs
    • n= 6,4096 base pairs
    • n= 8,65.5 kb base pairs

15. 16. Still Lots of Fragments

  • 4 cutter:3,000,000,000 bp
  • ~256 bp=12,000,000 fragments
  • 6 cutter:3,000,000,000 bp
  • ~4096 bp=700,000 fragments
  • 8 cutter:3,000,000,000 bp
  • ~65.5 kb=46,000 fragments

17. Ligation

  • sticky ends with complementary base pairs can form hydrogen bonds,
  • DNA ligase:an enzyme that catalyzes the reformation of the phosphodiester bonds.

18. Ligation 5 -------GA-A-T-T-C--------3 3 -------C-T-T-A-AG--------5 hydrogen bonds align, DNA ligase covalently links 19. DNA is Profligate

  • sticky ends with complementary base pairs can form hydrogen bonds,
  • with DNA fromanyspecies.

20. Cloning specialized DNA technology to produce multiple, exact copies of a single gene or other segment of DNA to obtain enough material for further study. 21.

  • Requires a Vector
  • ...a specialized DNA sequence that can enter a living cell,
  • ...signal its presence to an investigator,
  • ... and provide a means of replication for itself and the foreign DNA it carries.

Cloning specialized DNA technology to produce multiple, exact copies of a single gene or other segment of DNA to obtain enough material for further study. 22. And, Vectors

  • contain unique restriction sites to facilitate the creation of recombinant DNA molecules,
  • ... must also possess a distinguishing physical characteristic such as size or shape by which it can be purified away from the host cells genome.

Mike 23. Vectors

  • Plasmid E. coli up to 15 kb,
  • Phage E. coli up to 25 kb,
  • Cosmid E. coli up to 45 kb,
  • BAC E. coli 100-500 kb,
  • YAC Yeast 250-1000 kb.

24. Step 1restriction digests and ligation of fragments into cloning vectors, 25. Cloning Step 2

  • vector-insert recombinants are inserted in host cells,
    • Transformation (via bacterial mechanisms);
      • plasmids
      • BACS
      • YACS
    • Transduction (via virus mechanisms);
      • phage
      • cosmids

26. Cloning Step 3

  • ...vectors contain selectable markers,
    • only cells that contain vector DNA will survive selection,
    • recombinant vectors can be discerned from empty vectors by additional markers.

Blue/White Cloning 27. Amplification

  • if you were to grind me up with a giant mortar and pestle, and extract all of my DNA coding for a single gene, youd get about 1 g of DNA,
  • two liters of bacterial culture carrying my hemoglobin in a recombinant DNA vector, would yield an equivalent mass of DNA.

28. Assignment

  • Read 10.1 and be prepared to clone a fragment of DNA using a plasmid as a vector.

29. B-PCRvsA-PCR

  • PCR,P olymerasC hainR eaction

30. Today MoreDNA Science

  • DNA Amplification II,
    • Polymerase Chain Reaction ,
    • 6.7, pp. 237-239,
  • Gel Electrophoresis,
    • Southern Blots, Northern Blots,
    • 6.6, pp. 237-238,
  • Clones and Libraries, pp

31. P olymeraseC hainR eaction

  • invented by Kary Mullis while cruising in a Honda Civic on Highway 128 from San Francisco to Mendocino,
  • "It was quiet and something just went, Click!"

Kary B. Mullis Nobel Laureate, 1993 Chemistry 32.

  • " THE SUN HAD been hot that day in Mendocino County. A dry wind had come out of the east, and nobody knew how hot it had been until, around sunset, the wind stopped. I drove up from Berkeley through Cloverdale headed to Anderson Valley. The California buckeyes poked heavy blossoms out into Highway 128. The pink and white stalks hanging down into my headlights looked cold, but they were loaded with warmed oils that dominated the dimension of smell.
  • It seemed to be the night of the buckeyes, but something else was stirring.""My little silver Honda's front tires pulled us through the mountains. My hands felt the road and the turns. My mind drifted back to the lab. DNA chains coiled and floated. Lurid blue and pink images of electric molecules injected themselves somewhere between the mountain road and my eyes."

Opening words,Dancing Naked in the Mind Field , 1998, byDr.KaryMullis , Pantheon Books. 33. Mullis

  • ... PCR is a chemical procedure that will make the structures of the molecules of our genes as easy to see as billboards in the desert and as easy to manipulate as Tinkertoys.

DNA 34. Making DNA: Components test tube nucleus Environment ? DNA polIII DNA Polymerase ? primase Primer present present dNTPs ? helicase, etc. ss DNA template PCR Cell 35. Oligonucleotides specific primers

  • ...short pieces of synthetic DNA can be manufactured that contain any sequence,
  • template specific!

~ Odds of a Specific Sequence 20-mer:9.1 x 10 -13 36. Making One StrandOf DNA Add Polymerase Add dNTPs, etc. add primer 37. Making Two More Strands Must Denature Separate Strands 38. Denaturing cant use helicase in vitro

  • DNA denaturing conditions such as high heat or low salt concentrations irreversibly denature or inactivate most polymerases,
  • dNTPs are not affected by denaturation,
  • primers are not affected by denaturation.

39. Making Two More Strands Add polymerase, etc. add primer to second strand 40. Denaturation Step Bad

  • several rounds ofin vitroreplication could be performed (prior to PCR), however, accumulation of denatured polymerases quickly poisoned the reactions.

41.

  • bacteria discovered in a hot spring in Yellowstone Natural Park in 1965,
  • lives in salty water that ranges from 70 o- 75 oC,
  • thus, does DNA replication at very high temperatures.

42. Thermus aquaticusEnzymes

  • basic research demonstrated that many enzymes isolated fromThermus aquaticusfunction atveryhigh temperatures,
  • temperatures nearing 100 oC,
  • DNA denaturating temperatures.

43. Click

  • Kary Mullis realized that repetitive rounds of DNA synthesis could be performed by using a heat-stable polymerase,
  • T hermusaq uaticus :Taqpolymerase.

44. 94 o ~60 o 72 o Synthesis ~30 seconds/kb PCR 3--CGTACGTAATACGATGTAGCTGTAGCTGATCGTGAC--5 5--GCATGCATTAGGCTACATCGACATCGACTAGCACTG--3 5--GCATGCATTAT CTGATCGTGAC--5 Denature Step