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Manipulation of DNA

Manipulation of DNA. Polymerases - Needed for DNA and RNA synthesis - condensation synthesis of DNA or RNA: H 2 O produced - Phosphodiester bond binds nucleotide to existent strand of DNA/RNA - only polymerizes from 5 ’ to 3 ’ end, i.e. adding new nucleotides to the 3 ’ end.

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Manipulation of DNA

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  1. Manipulation of DNA Polymerases - Needed for DNA and RNA synthesis - condensation synthesis of DNA or RNA: H2O produced - Phosphodiester bond binds nucleotide to existent strand of DNA/RNA - only polymerizes from 5’ to 3’ end, i.e. adding new nucleotides to the 3’ end

  2. Polymerase Chain Reaction aim: amplify (i.e. get more copies) of a bit of DNA contained between 2 regions of known sequence.

  3. Polymerase Chain Reaction 5’ 3’ 5’ 3’ aim: amplify (i.e. get more copies) of a bit of DNA contained between 2 regions of known sequence. Requirements: - template: single stranded DNA or RNA

  4. Polymerase Chain Reaction 5’ 3’ 95°C 5’ 3’ aim: amplify (i.e. get more copies) of a bit of DNA contained between 2 regions of known sequence. Requirements: - template DNA DENATURATION

  5. Polymerase Chain Reaction 5’ 3’ 95°C 5’ 3’ aim: amplify (i.e. get more copies) of a bit of DNA contained between 2 regions of known sequence. Requirements: - template DNA - 1 pair of primers (short single-stranded fragment which will be complementary to the template and will allow the polymerase to start polymerising) DENATURATION 5’ 3’ 5’ 3’

  6. Polymerase Chain Reaction 5’ 3’ 95°C 5’ 3’ aim: amplify (i.e. get more copies) of a bit of DNA contained between 2 regions of known sequence. Requirements: - template DNA - 1 pair of primers DENATURATION 5’ 3’ 5’ 3’

  7. Polymerase Chain Reaction 5’ 3’ 95°C 5’ 3’ aim: amplify (i.e. get more copies) of a bit of DNA contained between 2 regions of known sequence. Requirements: - template DNA - 1 pair of primers DENATURATION 5’ 3’ 55°C 5’ 3’ ANNEALING OF PRIMERS

  8. Requirements (cted): - ThermostableTaQ Polymerase 5’ 3’ Polymerase Chain Reaction 5’ 3’

  9. Requirements (cted): - ThermostableTaQ Polymerase 5’ 3’ DNA polymerase Polymerase Chain Reaction 5’ 3’

  10. Requirements (cted): - ThermostableTaQ Polymerase - A supply of all 4 nucleotides 5’ 3’ DNA polymerase Polymerase Chain Reaction 5’ 3’ 5’ 3’ DNA polymerase DNA polymerase 5’ 3’ T T A A C C G G

  11. 5’ 3’ Requirements (cted): - ThermostableTaQ Polymerase DNA polymerase DNA polymerase Polymerase Chain Reaction DNA polymerase 5’ 3’

  12. Requirements (cted): - ThermostableTaQ Polymerase - A supply of all 4 nucleotides 5’ 3’ DNA polymerase DNA polymerase Polymerase Chain Reaction DNA polymerase 5’ 3’ 5’ 3’ DNA polymerase DNA polymerase DNA polymerase 5’ 3’ T T A A C C G G

  13. 5’ 3’ Requirements (cted): - A DNA polymerase C G Polymerase Chain Reaction 72°C DNA polymerase 5’ 3’ DNA EXTENSION T A C G

  14. 5’ 3’ Requirements (cted): - A DNA polymerase C G Polymerase Chain Reaction 72°C DNA polymerase 5’ 3’ DNA EXTENSION T A C G

  15. 5’ 3’ Requirements (cted): - thermocycler A DNA polymerase C G Polymerase Chain Reaction 72°C DNA polymerase 5’ 3’ DNA EXTENSION http://www.dnalc.org/resources/animations/pcr.html T A C G

  16. First cycle 95°C 55°C 72°C Denaturation Annealing DNA extension

  17. First cycle 95°C 55°C 72°C Denaturation Annealing DNA extension Second cycle

  18. First cycle 95°C 55°C 72°C Denaturation Annealing DNA extension Second cycle Third cycle 2x useful PCR products

  19. Each pair issued with: • Spare paper X1 • Row of primers pre-cut in bands. • First cycle template sheet • Pre-written products of second cycle • 1/ Teacher led: denaturation of DNA: cut with scissors as demo for all, then give them a template • For 1rst cycle of PCR, denatured DNA already in place. • 2/ Kids to glue their primers and extend by hand • 3/ Start of second cycle, kids to “denature” the products of the first cycle and do the gluing for • The next cycle on spare paper • 4/ The next cycle, then watch an animation • http://www.dnalc.org/resources/animations/pcr.html 5’ 3’ T A C G G A T T C G G G T A A C C G A A T G G C A T T G G A G A G G G A T A T G C C T A A G C C C A T T G G C T T A C C G T A A C C T C T C C C T A 5’ 3’

  20. C C G T C C G T C C G T C C G T C C G T C C G T C C G T C C G T C C G T C C G T C C G T C C G T C C G T C C G T C C G T C C G T C C G T C C G T C C G T C C G T C C G T C C G T C C G T C C G T C C G T C C G T C C G T C C G T C C G T C C G T C C G T C C G T C C G T C C G T C C G T C C G T C C G T C C G T C C G T C C G T C C G T C C G T C C G T C C G T C C G T T T C G T T C G T T C G T T C G T T C G T T C G T T C G T T C G T T C G T T C G T T C G T T C G T T C G T T C G T T C G T T C G T T C G T T C G T T C G T T C G T T C G T T C G T T C G T T C G T T C G T T C G T T C G T T C G T T C G T T C G T T C G T T C G T T C G T T C G T T C G T T C G T T C G T T C G T T C G T T C G T T C G T T C G T T C G T T C G T T C G

  21. First cycle of Polymerase chain reaction 5’ 3’ T A C G G A T T C G G G T A A C C G A A T G G C A T T G G A G A G G G A T A T G C C T A A G C C C A T T G G C T T A C C G T A A C C T C T C C C T A 5’ 3’ First cycle of Polymerase chain reaction 5’ 3’ T A C G G A T T C G G G T A A C C G A A T G G C A T T G G A G A G G G A T A T G C C T A A G C C C A T T G G C T T A C C G T A A C C T C T C C C T A 5’ 3’

  22. Polymerase Chain Reaction 5’ 3’ 95°C 5’ 3’ aim: amplify (i.e. get more copies) of a bit of DNA contained between 2 regions of known sequence. Requirements: - template DNA (Single stranded DNA or RNA) - 1 pair of primers (short single-stranded fragment which will be complementary to the template and will allow the polymerase to start polymerising) DENATURATION 5’ 3’ 55°C 5’ 3’ ANNEALING OF PRIMERS

  23. Requirements (cted): - Thermostable TaQ Polymerase ONLY POLYMERISES FROM 5’ end to 3’ end - A supply of all 4 nucleotides - a thermocycler (Obviously needed for the whole cycle) 5’ 3’ DNA polymerase DNA polymerase DNA polymerase 5’ 3’ 5’ 3’ DNA polymerase DNA polymerase DNA polymerase 5’ 3’ 5’ 3’ A DNA polymerase C G 72°C DNA polymerase T T T A A A 5’ 3’ C C C G G G DNA EXTENSION Repeat cycle x 20/30 and you get billions of the fragment to be amplified. Quantity of DNA doubles every cycle http://www.dnalc.org/resources/animations/pcr.html

  24. First cycle 95°C 55°C 72°C Denaturation Annealing DNA extension Second cycle Third cycle 2x useful PCR products

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