1 / 24

DNA Polymerase Manipulation and Polymerase Chain Reaction (PCR) Explained

This guide provides an overview of DNA polymerases and their role in DNA and RNA synthesis, including the condensation synthesis process with water production. It details the Polymerase Chain Reaction (PCR) method used to amplify specific DNA sequences between two known regions. Key requirements for successful PCR include a template (single-stranded DNA or RNA), a pair of primers for initiation, and the thermocycling process involving denaturation, annealing, and extension. Essential enzymes and conditions for effective DNA amplification are also highlighted.

evita
Télécharger la présentation

DNA Polymerase Manipulation and Polymerase Chain Reaction (PCR) Explained

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  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

More Related