Ampliqon seminar-pdf2

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End

End-point PCR Mark Møller

IRAN Distributor , Viragene Akam Co . Tel : (+9821) 88611552-3, 88062042-3

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AMPLIQON PCR Enzymes

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Overview

Short introduction to PCR AMPLIQON PCR Enzymes Most important optimization parameters Applications of AMPLIQON PCR Enzymes

Questions


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Taq DNA polymerase structure

Polymerase

Grey tones

DNA template

Blue

Primer

Red

Li et al., 1998 EMBO J. 17(24): 7514-25.


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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


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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


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DNA polymerases - Overview

DNA polymerases (Bacteria) Taq DNA polymerase

Tth DNA polymerase

DNA polymerases (Archaea) Pfu DNA polymerase

Pwo DNA polymerase


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Robust and reliable DNA polymerases DNA polymerases - Taq – TEMPase - AccuPOL

Master mixes

Specialized buffer systems for optimal performance

dNTP and additives


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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


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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


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Glycerol free DNA polymerases

Glycerol free Taq and Hot Start polymerases

Suitable for

Freeze drying

Automation processes for high throughput testing


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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


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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)


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AMPLIQON AccuPol DNA polymerase

HIGH FIDELITY – PROOFREADING

Cloning

Gene expression

Error rate 10-6

Processes up to 3 kb


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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


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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


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AMPLIQON PCR Buffer Systems

Standard Buffer – KCl

Ammomium Buffer – NH4+

Combination buffer – NH4+/KCl

Customized buffer solutions

GC buffer – optimized and additives


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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


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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


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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


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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 -


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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


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Optimization of PCR

1. Reaction components Mg2+ Template DNA Primer dNTP Enzyme

2. Temperature Denaturation Annealing Polymerisation

3. Time Denaturation Annealing Polymerisation + extra cycles


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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


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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


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Hot start technology increases specificity and yield

Specificity – Hot Start


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


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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


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PCR inhibitors

Source of inhibitors (Sample, extraction)

Inhibition ?

Dilution or further purification

Buffer

Additives

BSA (0.1-1.3 g/L)


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Applications


APPLICATION CHART x: recommended x: suitable

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ase

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Mix

K +

C

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+ C

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PO

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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


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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


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High throughput testing – Taq Master Mix Red

+ primers + PCR grade H2O

PCR

Load directly onto the gel


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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

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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.

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Quantification

TEMPase Hot Start

DNA polymerase

Ideal for qPCR applications

80ng – 0.08ng gDNA

High efficiency (98%)


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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


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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





PAH 12 primer gDNA

TEMPase Hot Start

Similar or better sensitivity compared to leading manufacturers


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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


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VWR PCR Enzymes

Questions?


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Health & Medicine

Ampliqon seminar-pdf2