Principles of PCR Small
-
Upload
muhammad-haris-lucky -
Category
Documents
-
view
225 -
download
0
Transcript of 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)
-
8/2/2019 Principles of PCR Small
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
-
8/2/2019 Principles of PCR Small
3/26
Structure of Nucleotide
5
3
-
8/2/2019 Principles of PCR Small
4/26
DNA Replication in vivo
-
8/2/2019 Principles of PCR Small
5/26
Phosphodiester Bond
5
3
DNA Synthesizing direction
-
8/2/2019 Principles of PCR Small
6/26
DNA of Organism
Gene 2
Gene 1
Gene 1 DNADNA
Copy of Gene 1
-
8/2/2019 Principles of PCR Small
7/26
DNA of Organism
Gene 2
Gene 1
Gene 1 DNADNA
Gene 2
Clone the fragments to the plasmid
Restriction digestion
-
8/2/2019 Principles of PCR Small
8/26
Control plasmid without insert
Control plasmidWith insert
Cloning Experiment
Bleu & white colonies
-
8/2/2019 Principles of PCR Small
9/26
Or PCR Amplification of the Gene
Gene 2
Gene 1
Gene 1 DNADNA
Copy of Gene 1
Primer Forwards
Reverse Primer
-
8/2/2019 Principles of PCR Small
10/26
3 5
Primer New synthesized DNA strand
53
-
8/2/2019 Principles of PCR Small
11/26
The Principe of PCR
-
8/2/2019 Principles of PCR Small
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.
-
8/2/2019 Principles of PCR Small
13/26
Step 1
Step 2 Step 3
-
8/2/2019 Principles of PCR Small
14/26
The Agarose Gel Electrophoreses ofDNA.
- Negative
+ Positive
DNA Fragments
-
8/2/2019 Principles of PCR Small
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
-
8/2/2019 Principles of PCR Small
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.
-
8/2/2019 Principles of PCR Small
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
-
8/2/2019 Principles of PCR Small
18/26
-
8/2/2019 Principles of PCR Small
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.
-
8/2/2019 Principles of PCR Small
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
-
8/2/2019 Principles of PCR Small
21/26
Template DNA
Cycle 1
Cycle 2
-
8/2/2019 Principles of PCR Small
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.
-
8/2/2019 Principles of PCR Small
23/26
DNA Directed RNA polymerase.
RNA Directed RNA polymerase (Virus enzyme).
RNA directed DNA Polymerase Reverse transcriptase(Virus enzyme).
-
8/2/2019 Principles of PCR Small
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
-
8/2/2019 Principles of PCR Small
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
-
8/2/2019 Principles of PCR Small
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