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