Principles of PCR Small

8/2/2019 Principles of PCR Small

1/26

Principle of PCR

(Polymerase Chain Reaction)

Dr. Farhad M. Abdulkarim BarzinjiDean of KMRC (Kurdistan Medical Research Center / HMU KRG)


2/26

DNA (Double Strand )

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

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


3/26

Structure of Nucleotide

5

3


4/26

DNA Replication in vivo


5/26

Phosphodiester Bond

5

3

DNA Synthesizing direction


6/26

DNA of Organism

Gene 2

Gene 1

Gene 1 DNADNA

Copy of Gene 1


7/26

DNA of Organism

Gene 2

Gene 1

Gene 1 DNADNA

Gene 2

Clone the fragments to the plasmid

Restriction digestion


8/26

Control plasmid without insert

Control plasmidWith insert

Cloning Experiment

Bleu & white colonies


9/26

Or PCR Amplification of the Gene

Gene 2

Gene 1

Gene 1 DNADNA

Copy of Gene 1

Primer Forwards

Reverse Primer


10/26

3 5

Primer New synthesized DNA strand

53


11/26

The Principe of PCR


12/26

Primers are synthetic ssDNA, can be synthesized orordered from company.

Length of the primers must not be less than 18 nts.

The GC and AT content of the 2 (reveres & forward) arefavored to be equal or closely.

Based on the length and GC / AT contain, the correcttemperature of hybridization of the primer to the targetDNA strand (annealing temp.) can be calculated.

Annealing temperature of the primers should be les than65 C, because of the extension temperature which is 73C, and that is due to the optimum temp of Taq-DNA

Polymerase which is 73 C.


13/26

Step 1

Step 2 Step 3


14/26

The Agarose Gel Electrophoreses ofDNA.

- Negative

+ Positive

DNA Fragments


15/26

Comparison between Wild and Mutated Plasmid

B AD CF EGH M1kb

Recombinant

plasmidMutated

plasmid

SalI ,BamHI

PvuII

BamHI

Control

BamHI

PvuII of r. p


16/26

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.


17/26

The PCR cocktail

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

The mixes

1) ddH2O xul

2) Buffer 1x

3) ddNTPs 1mm

4) Primers 10pmols

5) Template 1-10ng

6) Polymerase 0.5U


18/26


19/26

PCR Machine: The Segments & Parameters

Segment 1 rapid rising to Denaturing temperature

(94

0

C). 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.


20/26

Segment1

Segment 2Seg

men

t3

Segment 4S

egm

ent5

Segment 6 Seg

m

ent7

Segment 8

Denaturing temp. Annealing Temp Extension temp.prestart IncubationAt + 4 oC

Thermo cycle

5- 45 cycles


21/26

Template DNA

Cycle 1

Cycle 2


22/26

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

1

2

4

8

First cycle

Second cycle

Third cycle

16

Forth cycle & etc.


23/26

DNA Directed RNA polymerase.

RNA Directed RNA polymerase (Virus enzyme).

RNA directed DNA Polymerase Reverse transcriptase(Virus enzyme).


24/26

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

00

00

00


25/26

PCR Products for M (Matrix gene) Primer

Forward Primer Sequence5- ACCGAGGTCGAAACG 3-

13 ACCGAGGTCGAAACG 27

Reveres Primer Sequence5- AGGGCATTTTGGACAAAGCGTCTA 3-

251 AGGGCATTTTGGACAAAGCGTCTA 228

20

30

50

288-13=215 R. Primer

F. Primer

bp

bp


26/26

PCR Products for HA & NA Primer

Reveres Primer Sequence HA

5 CCCTCTTATCAACTCAACATAAAAACA 3

Forward Primer Sequence NA

5GGGTGATTGAGAAATGAATCCAAATCA 3

Reveres Primer Sequence HA

5 CGCGAGTAGAAACAAGGGTGTTTTT 3

Forward Primer Sequence HA

5CGCGCAGACCAAAAGCAGGGG 3

500

b

700

Documents

Principles of PCR Small