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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. - PowerPoint PPT Presentation
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Manipulation of DNAPolymerases
- 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
Polymerase Chain Reaction
- aim: amplify (i.e. get more copies) of a bit of DNA contained between 2 regions of known sequence.
Polymerase Chain Reaction
- 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
5’
5’
3’
3’
Polymerase Chain Reaction
- aim: amplify (i.e. get more copies) of a bit of DNA contained between 2 regions of known sequence.
- Requirements:- template DNA
5’
5’
3’
3’
DENATURATIONDENATURATION
Polymerase Chain Reaction
- 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)
5’
5’
3’
3’
DENATURATIONDENATURATION
5’
5’
3’
3’
Polymerase Chain Reaction
- 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
5’
5’
3’
3’
DENATURATIONDENATURATION
5’
5’
3’
3’
Polymerase Chain Reaction
- 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
5’
5’
3’
3’
DENATURATIONDENATURATION
5’
5’
3’
3’
ANNEALING OF PRIMERS
ANNEALING OF PRIMERS
Polymerase Chain Reaction
- Requirements (cted):- Thermostable TaQ
Polymerase5’
5’
3’
3’
Polymerase Chain Reaction
- Requirements (cted):- Thermostable TaQ
Polymerase
DNA polymerase
DNA polymerase
5’
5’
3’
3’
Polymerase Chain Reaction
DNA polymerase
DNA polymerase
5’
5’
3’
3’
5’
5’
3’
3’
DNA polymerase
DNA polymerase
DNA polymerase
DNA polymerase
A T
C G
A T
C G
- Requirements (cted):- Thermostable TaQ
Polymerase
- A supply of all 4 nucleotides
Polymerase Chain Reaction
DNA polymerase
DNA polymerase
- Requirements (cted):- Thermostable TaQ
Polymerase
5’
5’
3’
3’
DNA polymerase
DNA polymerase
DNA polymerase
DNA polymerase
Polymerase Chain Reaction
5’
5’
3’
3’
- Requirements (cted):- Thermostable TaQ
Polymerase
- A supply of all 4 nucleotides
DNA polymerase
DNA polymerase
DNA polymerase
DNA polymerase
5’
5’
3’
3’
DNA polymerase
DNA polymerase
DNA polymerase
DNA polymerase
A T
C G
A T
C G
DNA polymerase
DNA polymerase
DNA polymerase
DNA polymerase
Polymerase Chain Reaction
- Requirements (cted):-
5’
5’
3’
3’
DNA polymerase
DNA polymerase
DNA polymerase
DNA polymerase
A
C G
A T
C G
DNA EXTENSIONDNA EXTENSION
Polymerase Chain Reaction
- Requirements (cted):-
5’
5’
3’
3’
DNA polymerase
DNA polymerase
DNA polymerase
DNA polymerase
A
C G
A T
C G
DNA EXTENSIONDNA EXTENSION
Polymerase Chain Reaction
- Requirements (cted):- thermocycler
5’
5’
3’
3’
DNA polymerase
DNA polymerase
DNA polymerase
DNA polymerase
A
C G
A T
C G
DNA EXTENSIONDNA EXTENSION
http://www.dnalc.org/resources/animations/pcr.html
First cycle 95°C 55°C 72°C Denaturation Annealing DNA extension
First cycle 95°C 55°C 72°C Denaturation Annealing DNA extension
Second cycle
First cycle 95°C 55°C 72°C Denaturation Annealing DNA extension
Second cycle
Third cycle
2x usefulPCR products
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 templateFor 1rst cycle of PCR, denatured DNA already in place.2/ Kids to glue their primers and extend by hand3/ Start of second cycle, kids to “denature” the products of the first cycle and do the gluing forThe next cycle on spare paper 4/ The next cycle, then watch an animationhttp://www.dnalc.org/resources/animations/pcr.html
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
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
3’ 5’
3’5’
C C G T
T T C G
C C G T
T T C G
C C G T
T T C G
C C G T
T T C G
C C G T
T T C G
C C G T
T T C G
C C G T
T T C G
C C G T
T T C G
C C G T
T T C G
C C G T
T T C G
C C G T
T T C G
C C G T
T T C G
C C G T
T T C G
C C G T
T T C G
C C G T
T T C G
C C G T
T T C G
C C G T
T T C G
C C G T
T T C G
C C G T
T T C G
C C G T
T T C G
C C G T
T T C G
C C G T
T T C G
C C G T
T T C G
C C G T
T T C G
C C G T
T T C G
C C G T
T T C G
C C G T
T T C G
C C G T
T T C G
C C G T
T T C G
C C G T
T T C G
C C G T
T T C G
C C G T
T T C G
C C G T
T T C G
C C G T
T T C G
C C G T
T T C G
C C G T
T T C G
C C G T
T T C G
C C G T
T T C G
C C G T
T T C G
C C G T
T T C G
C C G T
T T C G
C C G T
T T C G
C C G T
T T C G
C C G T
T T C G
C C G T
T T C G
First cycle of Polymerase chain reaction
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 3’5’
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
3’ 5’First cycle of Polymerase chain reaction
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 3’5’
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
3’ 5’
Polymerase Chain Reaction
- 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)
5’
5’
3’
3’
DENATURATIONDENATURATION
5’
5’
3’
3’
ANNEALING OF PRIMERS
ANNEALING OF PRIMERS
5’
5’
3’
3’
- Requirements (cted):- Thermostable TaQ
PolymeraseONLY POLYMERISES FROM 5’ end to 3’
end
- A supply of all 4 nucleotides
- a thermocycler (Obviously needed for the whole cycle)
DNA polymerase
DNA polymerase
DNA polymerase
DNA polymerase
5’
5’
3’
3’
DNA polymerase
DNA polymerase
DNA polymerase
DNA polymerase
A T
C G
A T
C G
DNA polymerase
DNA polymerase
DNA polymerase
DNA polymerase
5’
5’
3’
3’
DNA polymerase
DNA polymerase
DNA polymerase
DNA polymerase
A
C G
A T
C G
DNA EXTENSIONDNA 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
First cycle 95°C 55°C 72°C Denaturation Annealing DNA extension
Second cycle
Third cycle
2x usefulPCR products