Principle of PCR (Polymerase Chain Reaction)

Dr. Farhad M. Abdulkarim Barzinji

Dean of KMRC (Kurdistan Medical Research Center / HMU KRG)

DNA (Double Strand )

5 A 3 T

A G T C G T A A T 3 T C A G C A T T A 5

Structure of Nucleotide

DNA Replication in vivo

Phosphodiester Bond

5 DNA Synthesizing direction

DNA of Organism

DNA

Gene 1

Gene 2

DNA

Copy of Gene 1

Gene 1

DNA of Organism
Restriction digestion

DNA

Gene 1

Gene 2

DNA

Gene 2 Gene 1

Clone the fragments to the plasmid

Bleu & white colonies

Control plasmid With insert

Control plasmid without insert

Cloning Experiment

Or PCR Amplification of the Gene

Primer Forwards

DNA

Gene 1

Gene 2

DNA Reverse Primer

Copy of Gene 1

Gene 1

3 5 Primer

5 3 New synthesized DNA strand

The Principe of PCR

 Primers are synthetic ssDNA, can be synthesized or ordered from company. Length of the primers must not be less than 18 nts. The GC and AT content of the 2 (reveres & forward) are favored to be equal or closely. Based on the length and GC / AT contain, the correct temperature of hybridization of the primer to the target DNA strand (annealing temp.) can be calculated. Annealing temperature of the primers should be les than 65 C, because of the extension temperature which is 73 C, and that is due to the optimum temp of Taq-DNA Polymerase which is 73 C.

Step 1

Step 2

Step 3

The Agarose Gel Electrophoreses of DNA.

- Negative

DNA Fragments

+ Positive

Comparison between Wild and Mutated Plasmid

H G F E D C B A M 1kb

Mutated plasmid PvuII Control BamHI SalI , BamHI

Recombinant plasmid

BamHI

PvuII of r. p

PCR (Polymerase Chain Reaction)

Components for PCR reaction: a) Template b) Taq DNA polymerase c) dNTPs (dATP, dCTP, dGTP, dTTP) d) Buffer + Mg e) ddH2O. f) 2 Primers (forward & reverse) g) PCR machine.

The PCR cocktail

The volume of PCR cocktail can be either 25ul 50ul 100ul.

1) 2) 3) 4) 5) 6)

The mixes
ddH2O xul Buffer 1x ddNTPs 1mm Primers 10pmols Template 1-10ng Polymerase 0.5U

PCR Machine: The Segments & Parameters Segment 1 rapid rising to Denaturing temperature (940C). Segment 2 Denaturing temperature (940C) Segment 3 rapid reducing to the desired annealing temperature (based on primers) Segment 4 annealing temperature. Segment 5 rapid rising to extension temperature. Segment 6 extension temperature. Segment 7 +40C.

Segment 2
Se g m ent 5

nt 3 me S eg

Segm en

Segment 6

t1

ent 7 Segm

Segment 4

Segment 8 prestart Denaturing temp. Annealing Temp Extension temp. Incubation At + 4 oC

Thermo cycle 5- 45 cycles

Template DNA Cycle 1

Cycle 2

PCR Reaction (number of cycles are upon request 5-45 cycles)

1
First cycle

2
Second cycle

4
Third cycle

8
Forth cycle & etc. 16

 DNA Directed RNA polymerase. RNA Directed RNA polymerase (Virus enzyme). RNA directed DNA Polymerase Reverse transcriptase (Virus enzyme).

Complimentary DNA (cDNA)

Total RNA extraction from the cell. Primer directed to a sequence of the RNA ( Poly A Tail sequence) e.g. Poly T primer.

RNA cDNA

Poly A Tail Primer Poly T

200

300

Forward Primer Sequence

5- ACCGAGGTCGAAACG 313 ACCGAGGTCGAAACG 27

Reveres Primer Sequence

5- AGGGCATTTTGGACAAAGCGTCTA 3251 AGGGCATTTTGGACAAAGCGTCTA 228

288-13=215

500

R. Primer F. Primer

PCR Products for M (Matrix gene) Primer

500 bp

Forward Primer Sequence HA 5CGCGCAGACCAAAAGCAGGGG 3 Reveres Primer Sequence HA 5 CGCGAGTAGAAACAAGGGTGTTTTT 3

Forward Primer Sequence NA 5GGGTGATTGAGAAATGAATCCAAATCA 3 Reveres Primer Sequence HA 5 CCCTCTTATCAACTCAACATAAAAACA 3

PCR Products for HA & NA Primer

700 b