Upload
maziar-yari
View
204
Download
1
Embed Size (px)
Citation preview
1
End
End-point PCR Mark Møller
IRAN Distributor , Viragene Akam Co . Tel : (+9821) 88611552-3, 88062042-3
WWW.AMPLIQON.IR .
2
AMPLIQON PCR Enzymes
1
Overview
Short introduction to PCR AMPLIQON PCR Enzymes Most important optimization parameters Applications of AMPLIQON PCR Enzymes
Questions
IRAN Distributor , Viragene Akam Co . Tel : (+9821) 88611552-3, 88062042-3
WWW.AMPLIQON.IR .
3
Taq DNA polymerase structure
Polymerase
Grey tones
DNA template
Blue
Primer
Red
Li et al., 1998 EMBO J. 17(24): 7514-25.
IRAN Distributor , Viragene Akam Co . Tel : (+9821) 88611552-3, 88062042-3
WWW.AMPLIQON.IR .
4
DNA
Double-stranded DNA arranged in a double helix DNA strand
• a sugar-phosphate backbone
• the 4 bases adenine, guanine, cytosine and thymine
The 4 bases come in pairs
• A – T • G – C
The DNA double helix
sugar
phosphate
backbone
hydrogen
bond
base
4
IRAN Distributor , Viragene Akam Co . Tel : (+9821) 88611552-3, 88062042-3
WWW.AMPLIQON.IR .
5
PCR = Polymerase Chain Reaction PCR ”Molecular photocopying”
ds DNA 95 °C melting
50 - 60 °C annealing
72 °C extension
PCR cycle 1 PCR cycle 2 PCR cycle 3
PCR cycle 4
95 °C, 55 °C 72 °C
95 °C
50 - 60 °C
72 °C 95 °C
50 - 60 °C
72 °C
40 °C
100 °C
Temperature profile:
… PCR cycle 30
IRAN Distributor , Viragene Akam Co . Tel : (+9821) 88611552-3, 88062042-3
WWW.AMPLIQON.IR .
6
DNA polymerases - Overview
DNA polymerases (Bacteria) Taq DNA polymerase
Tth DNA polymerase
DNA polymerases (Archaea) Pfu DNA polymerase
Pwo DNA polymerase
IRAN Distributor , Viragene Akam Co . Tel : (+9821) 88611552-3, 88062042-3
WWW.AMPLIQON.IR .
7
Robust and reliable DNA polymerases DNA polymerases - Taq – TEMPase - AccuPOL
Master mixes
Specialized buffer systems for optimal performance
dNTP and additives
IRAN Distributor , Viragene Akam Co . Tel : (+9821) 88611552-3, 88062042-3
WWW.AMPLIQON.IR .
8
Taq DNA polymerase
First heat stable enzyme for PCR
Thermophile Thermus aquaticus
Processes up to 5 kb
5´-3´DNA polymerase and exonuclease activity
No 3´-5´exonuclease activity (no proofreading)
High extension rate (40-100 nucleotides/sec at 72°C)
High processivity (~60 nt)
Leaves A overhang
Keep on ice when mixed with primers and template
IRAN Distributor , Viragene Akam Co . Tel : (+9821) 88611552-3, 88062042-3
WWW.AMPLIQON.IR .
9
AMPLIQON Taq DNA Polymerase
A good choice for routine applications
Reliable and high yield DNA amplification
Quality control of each batch
Stable at 25°C for >8 months
Up to 50 freeze-thaw cycles
Shelf life 3 years
Affordable price
IRAN Distributor , Viragene Akam Co . Tel : (+9821) 88611552-3, 88062042-3
WWW.AMPLIQON.IR .
10
Glycerol free DNA polymerases
Glycerol free Taq and Hot Start polymerases
Suitable for
Freeze drying
Automation processes for high throughput testing
IRAN Distributor , Viragene Akam Co . Tel : (+9821) 88611552-3, 88062042-3
WWW.AMPLIQON.IR .
11
PFU DNA polymerase
Hyperthermophile Pyrococcus furiosus
5´-3´DNA polymerase activity
3´-5´exonuclease activity (proofreading)
Extension rate ~ 25 nucleotides/sec at 72°C
Processivity ~ 20 nucleotides
Blunt ended DNA
Applications
Cloning and gene expression
5’
5’
3’
3’
New strand grows in 5´-3´direction
polymerase
IRAN Distributor , Viragene Akam Co . Tel : (+9821) 88611552-3, 88062042-3
WWW.AMPLIQON.IR .
12
Fidelity/error rates DNA pol. fidelity: ratio of wrong to correct nucleotide
incorporations
Fidelity depends on the polymerase, the buffer, and the quality of the DNA
Fidelity value ranges Taq 10-5 (1 mistake per app. 50.000 bp)
Proofreaders 10-6 (1 mistake per app. 500.000 bp)
IRAN Distributor , Viragene Akam Co . Tel : (+9821) 88611552-3, 88062042-3
WWW.AMPLIQON.IR .
13
AMPLIQON AccuPol DNA polymerase
HIGH FIDELITY – PROOFREADING
Cloning
Gene expression
Error rate 10-6
Processes up to 3 kb
IRAN Distributor , Viragene Akam Co . Tel : (+9821) 88611552-3, 88062042-3
WWW.AMPLIQON.IR .
14
Modified DNA polymerases
Hot start DNA polymerases Antibody (1-5 min. at 95°C)
Chemical (10-15 min. at 95°C)
Aptamer (< 1 min. at 95°C)
Not active at room temperature
Reduces non-specific priming and primer-dimers
Higher specificity and sensitivity
IRAN Distributor , Viragene Akam Co . Tel : (+9821) 88611552-3, 88062042-3
WWW.AMPLIQON.IR .
15
TEMPase Hot Start DNA polymerase
Higher sensitivity and specificity
Chemically modified
Reaction setup at room temperature
Activated by initial heating, 15 min at 95°C
+Hot Start
Without Hot Start
IRAN Distributor , Viragene Akam Co . Tel : (+9821) 88611552-3, 88062042-3
WWW.AMPLIQON.IR .
16
AMPLIQON PCR Buffer Systems
Standard Buffer – KCl
Ammomium Buffer – NH4+
Combination buffer – NH4+/KCl
Customized buffer solutions
GC buffer – optimized and additives
IRAN Distributor , Viragene Akam Co . Tel : (+9821) 88611552-3, 88062042-3
WWW.AMPLIQON.IR .
17
PCR Buffer Systems
Buffer
Features Standard Ammonium Combination
High yield √ √√ √
High specificity √√ √ √√
Tolerance for primer annealing temperature
÷ √ √
M
S A
ENG5
Standard buffer Ammonium buffer Combination buffer
Ammonium buffer is the first choice
IRAN Distributor , Viragene Akam Co . Tel : (+9821) 88611552-3, 88062042-3
WWW.AMPLIQON.IR .
18
RED AND BLUE Master Mixes Easy to use
Add template, primers and press RUN
Ready to use in agarose gel electrophoresis
No need for additional sample loading buffer
TEMPase Hot Start Master Mix BLUE
Taq DNA Polymerase Master Mix RED
1 1 N 2 2 N M
IRAN Distributor , Viragene Akam Co . Tel : (+9821) 88611552-3, 88062042-3
WWW.AMPLIQON.IR .
19
dNTP ≥99% purity
Highly stable
Ready-to-use molecular grade dNTP
dNTP set (dATP, dCTP, dGTP and dTTP, 100mM)
dNTP mixes (100mM, 40mM)
DNA polymerisation
DNA labelling
Sequencing processes
IRAN Distributor , Viragene Akam Co . Tel : (+9821) 88611552-3, 88062042-3
WWW.AMPLIQON.IR .
20
Setting up the PCR master mix
Reaction components
Always include negative (and positive) controls
Component Vol./reaction Final concentration
10x Buffer (15 mM MgCl2) 5 µl 1x
25 mM MgCl2 0 µl (0 – 7 µl) 1.5 mM (0.5 – 5 mM)
dNTP mix (12.5 mM each) 0.8 µl 0.2 mM of each dNTP
Primer A (10 µM) 1 µl (0.5 – 5 µl) 0.2 µM (0.1 – 1.0 µM)
Primer B (10 µM) 1 µl (0.5 – 5 µl) 0.2 µM (0.1 – 1.0 µM)
Taq DNA Polymerase 0.2 µl (0.2 – 1 µl) 2 units (1 – 5 units)
PCR-grade H2O X µl -
Template DNA X µl
genomic DNA: 50 ng (10 – 500 ng) plasmid DNA: 0.5 ng (0.1 – 1 ng)
bacterial DNA: 5 ng (1 – 10 ng)
TOTAL volume 50 µl -
IRAN Distributor , Viragene Akam Co . Tel : (+9821) 88611552-3, 88062042-3
WWW.AMPLIQON.IR .
21
Optimization of end-point PCR
My PCR works without any further optimization!
Specificity is important
And also sensitivity
M 20ng 5ng 1.25ng 0.3ng 80pg 20pg 5pg 1.25pg 0.3pg ntc
TEMPase in Ammonium buffer
IRAN Distributor , Viragene Akam Co . Tel : (+9821) 88611552-3, 88062042-3
WWW.AMPLIQON.IR .
22
Optimization of PCR
1. Reaction components Mg2+ Template DNA Primer dNTP Enzyme
2. Temperature Denaturation Annealing Polymerisation
3. Time Denaturation Annealing Polymerisation + extra cycles
IRAN Distributor , Viragene Akam Co . Tel : (+9821) 88611552-3, 88062042-3
WWW.AMPLIQON.IR .
23
Mg2+ optimization increases specificity
0.5-4.5 mM Mg2+
dNTP, EDTA
Yield vs. specificity
Specificity – Mg2+
M DMD single bands and 10-plex M
IRAN Distributor , Viragene Akam Co . Tel : (+9821) 88611552-3, 88062042-3
WWW.AMPLIQON.IR .
24
Optimization of annealing temperature increases specificity
Temp. gradient 51-66°C
Specificity increases with increasing temperature
Specificity – Annealing temperature
51 54 57 60 63 66
IRAN Distributor , Viragene Akam Co . Tel : (+9821) 88611552-3, 88062042-3
WWW.AMPLIQON.IR .
25
Hot start technology increases specificity and yield
Specificity – Hot Start
M DMD single bands and 10-plex M
M 0.5 1 1.5 2 2.5 3 3.5 4 4.5
mM magnesium
M 0.5 1 1.5 2 2.5 3 3.5 4 4.5
mM magnesium
Taq
TEMPase
IRAN Distributor , Viragene Akam Co . Tel : (+9821) 88611552-3, 88062042-3
WWW.AMPLIQON.IR .
26
PCR inhibitors
Type Compound/ion Source Mechanism
DNA polymerase inhibitors
Bile salts Faeces Direct effect on polymerase
Ca2+ Bone, milk Competition with Mg2+
EDTA Anticoagulant Chelation of Mg2+
Haem/haemoglobin Blood Lowers polymerase processivity
Phenol DNA purification Binding to polymerase
Nucleic acid inhibitors
Humic acids Soil, bone Binds to nucleic acids
Collagen Bone Lowers polymerase processivity by binding to DNA
Ethanol DNA extraction Precipitation of DNA
Formaldehyde Preservative Interference with DNA
Melanin Skin, hair Binds to DNA
Source: J. Hedman, Phd thesis, DNA Analysis of PCR-Inhihitory Forensic Samples
IRAN Distributor , Viragene Akam Co . Tel : (+9821) 88611552-3, 88062042-3
WWW.AMPLIQON.IR .
27
PCR inhibitors
Source of inhibitors (Sample, extraction)
Inhibition ?
Dilution or further purification
Buffer
Additives
BSA (0.1-1.3 g/L)
IRAN Distributor , Viragene Akam Co . Tel : (+9821) 88611552-3, 88062042-3
WWW.AMPLIQON.IR .
28
Applications
M DMD single bands and 10-plex M
APPLICATION CHART x: recommended x: suitable
Ta
q D
NA
Po
lym
eras
e
G
lyce
rol-
fre
e
Ta
q D
NA
Po
lym
eras
e
R
ED T
aq D
NA
Po
lym
eras
e
Ta
q D
NA
Po
lym
eras
e
M
aste
r M
ix
R
ED Ta
q D
NA
Po
lym
eras
e
M
aste
r M
ix
TE
MP
ase
Ho
t St
art
DN
A P
oly
mer
ase
G
lyce
rol-
fre
e T
EMP
ase
Ho
t St
art
DN
A P
oly
mer
ase
TE
MP
ase
Ho
t St
art
Mas
ter
Mix
K +
C
B
lue
TEM
Pa
se H
ot
Star
t
M
aste
r M
ix K
+ C
G
C-r
ich
DN
A T
arge
t ki
t
G
C T
EMP
ase
Mas
ter
Mix
I +
II
M
ult
iple
x TE
MP
ase
Mas
ter
Mix
A
ccu
PO
L D
NA
Po
lym
eras
e
Application Standard PCR Hot Start Special PCR Hi Fi
Routine PCR x x x x x x x
High throughput x x x x x x x x x
Automation x x
GC-rich DNA templates x x x
Multiplex PCR x x
Sequencing x
Genotyping x x x x x x x x x x
Cloning x x x
Mutagenesis x
Freeze-drying x x
Low abundance targets x x x x x x x
Forensics x
DNA fingerprinting x
Colony PCR x x x x x x x x x x x
Gene expression x x x x x x
Microbial/Pathogen detection x x x x x x x
Quantitation x
IRAN Distributor , Viragene Akam Co . Tel : (+9821) 88611552-3, 88062042-3
WWW.AMPLIQON.IR .
29
Multiplex Master Mix
Multiplex PCR
700bp
300bp
M CFTR 5-plex
200bp
500bp
Applications:
Detection and typing of microorganisms
Genotyping
DMD single bands 10-plex M
IRAN Distributor , Viragene Akam Co . Tel : (+9821) 88611552-3, 88062042-3
WWW.AMPLIQON.IR .
30
High throughput testing – Taq Master Mix Red
+ primers + PCR grade H2O
PCR
Load directly onto the gel
IRAN Distributor , Viragene Akam Co . Tel : (+9821) 88611552-3, 88062042-3
WWW.AMPLIQON.IR .
31
GC-rich DNA amplification
Guanine Cytosine
Adenine Thymine
hydrogen bonds PCR failure – High GC content of template?
Hydrogen bonds bind the two strands in doublestranded DNA
G≡C 3 hydrogen bonds A꞊T 2 hydrogen bonds More energy is required to separate GC
than AT
Higher temperature and/or additives
31
IRAN Distributor , Viragene Akam Co . Tel : (+9821) 88611552-3, 88062042-3
WWW.AMPLIQON.IR .
32
GC-rich DNA amplification
GC Buffer I or II and use TEMPase excellent amplification of GC-rich DNA
Six genes with a varying percentage of GC contents were amplified with different buffers
One buffer type is not enough
M ENG9 CDK5 ENG5 KLF14 ABCC8 FECH1 M % GC: 58.4 64.2 68.5 71.4 72.9 76.6
Buffer: S A I II S A I II S A I II S A I II S A I II S A I II
S: Standard Buffer, A: Ammonium Buffer, I: GC Buffer I, II: GC Buffer II and M: marker.
32
IRAN Distributor , Viragene Akam Co . Tel : (+9821) 88611552-3, 88062042-3
WWW.AMPLIQON.IR .
33
Quantification
TEMPase Hot Start
DNA polymerase
Ideal for qPCR applications
80ng – 0.08ng gDNA
High efficiency (98%)
IRAN Distributor , Viragene Akam Co . Tel : (+9821) 88611552-3, 88062042-3
WWW.AMPLIQON.IR .
34
Product comparisons hot start competitors
ENG5
AMPL Q L T N
PAH12
AMPL Q L T N
Q8N4S7
AMPL Q L T N
ENG9
AMPL Q L T N
4 primer pairs gDNA
TEMPase Hot start
just as good or better compared to leading manufacturers
IRAN Distributor , Viragene Akam Co . Tel : (+9821) 88611552-3, 88062042-3
WWW.AMPLIQON.IR .
35
Product comparisons sensitivity
AMPLIQON Chemical
20 ng 5 ng 1.25 ng 0.3 ng 0.08 ng 0.02 ng 5 pg 1.25 pg 0.3 pg NTC
Competitor Q Chemical
Competitor L Chemical
Competitor N Aptamer
Competitor T Antibody
20 ng 5 ng 1.25 ng 0.3 ng 0.08 ng 0.02 ng 5 pg 1.25 pg 0.3 pg NTC
20 ng 5 ng 1.25 ng 0.3 ng 0.08 ng 0.02 ng 5 pg 1.25 pg 0.3 pg NTC
20 ng 5 ng 1.25 ng 0.3 ng 0.08 ng 0.02 ng 5 pg 1.25 pg 0.3 pg NTC
20 ng 5 ng 1.25 ng 0.3 ng 0.08 ng 0.02 ng 5 pg 1.25 pg 0.3 pg NTC
PAH 12 primer gDNA
TEMPase Hot Start
Similar or better sensitivity compared to leading manufacturers
IRAN Distributor , Viragene Akam Co . Tel : (+9821) 88611552-3, 88062042-3
WWW.AMPLIQON.IR .
36
AMPLIQON PCR Enzymes
Next week please remember
AMPLIQON PCR Enzymes are good choices for PCR
The enzymes are highly temperature stable
Buffers for different applications
You can receive samples of all the products
• Take a look in the AMPLIQON PCR Enzymes catalogue
IRAN Distributor , Viragene Akam Co . Tel : (+9821) 88611552-3, 88062042-3
WWW.AMPLIQON.IR .