modified for use in "Hot Start" PCR
specific DNA amplification
time and effort
in set-up times do not affect reproducibility
DNA Polymerase is used in "Hot Start" PCR, a procedure shown
to improve specificity and product yield. SurePRIME is heat-activated
after a preincubation step at 95°C and is then functionally equivalent
to classical Taq DNA Polymerase.
modified recombinant Taq DNA polymerase
The enzyme is a highly purified form of recombinant Taq polymerase that
has been chemically modified by the addition of heat-labile blocking groups
to specific amino acid residues. Prior to PCR, in its inactive state SurePRIME is incapable of extending primer-dimers or mis-annealed primer-template
species that form below the specific annealing temperature. The 95°C
incubation step therefore serves to activate the enzyme and also to ensure
a completely "clean" initial PCR cycle.
Once activated SurePRIME is functionally equivalent to Taq
DNA Polymerase and so the only PCR program modification required is the
addition of a pre-incubation step at 95°C. Alternatively, a linear
activation protocol may be used.
time and effort
Switching to SurePRIME avoids wasting time with complicated
and time-consuming manual "hot start" procedures or less efficient
under challenging PCR conditions
The performance of SurePRIMEand classical recombinant Taq DNA
polymerase was compared under challenging PCR conditions. A 400 bp
region of the human ß-globin gene was amplified using specially
designed non-optimized primers. The primer set contained mismatched
bases at the 5' end. As shown above, Taq polymerase was not capable
of providing a specific amplification whereas only the expected 400
bp fragment was amplified with SurePRIME DNA Polymerase.
this experiment amplification conditions favouring the formation
of non-specific product were purposely employed non-optimized
primers and 30 minutes pre-incubation at room temperature. For details
see Figure 1 & 2.
to demonstrate performance of SurePRIME with non-optimized primers
A set of non-optimized primers amplifying a 400 bp region of the human
ß-globin gene was made. The primer set (see below) was designed
with mismatched bases at the 5' end to represent PCR conditions frequently
encountered in cloning (e.g. introduction of a restriction site). Two
parallel sets of experiments were set up using SurePRIME and classical
Taq DNA polymerase. PCR mixes were made using the recommended buffer
for each enzyme and 100ng of template DNA.
influence the conditions for nonspecific priming, the PCR reactions, once
set up, were incubated for 30 min at 25 °C. Under these conditions
classical Taq DNA polymerase exhibits some polymerase activity,
enough to allow elongation of the mismatched primers. Following this incubation
step, the "Hot Start" enzyme was activated by heating for 15
min at 95 °C and then the same PCR programme was used for both sets
5 CTG GCC ACT CAC CTT AGG GT
Tm = 36 °C for specific bases (black) 60°C for whole oligo
- 20 nts
5 TCG CAG TTC TGG GCA TAA AAG
Tm = 34 °C for specific bases (black) 58 °C for whole oligo
- 21 nts
[30 à 25°C] [(5 at 93°C) x 1]
- [(1 at 45°C 2 at 70°C
1 at 93°C) x 37] - [(1 at 45°C 10
at 70°C) x 1] Hold at 30°C
[30 at 25°C] [(15 at 95°C) x 1] - [(1
at 45°C 2 at 70°C
1 at 93°C) x 37] [(1 at 45°C 10
at 70°C) x ] Hold at 30°C
Lane 1: 1U
Lane 2: 2U
Lane 3: 5 U of Taq DNA polymerase, respectively with 100 ng template
Lane 4: pBR322/HaeIII
Lane 1: 1U
Lane 2: 2U
Lane 3: 5 U of SurePRIME"Hot Start" polymerase, respectively
with 100 ng template DNA.
Lane 4: pBR322/HaeIII.
- The enzyme
exhibits no activity at room temperature. This means that variations
in the time taken to set up reactions will not affect reproducibility
- SurePRIMEDNA polymerase provides highly specific DNA amplification even when
using non-optimized primers.Unit definition
is the amount of enzyme required to catalyze the incorporation of 10 nanomoles
of nucleosides into a DE81 adsorbable product within 30 mm at 70°C
under assay condition.
20mM Tris-HCl pH 8.0, 100mM KCl,
0.1mM EDTA, 1mM dithiothreitol,
0.5% Tween 20, 0.5% Nonidet P40,
10mM Tris-HCl pH 8.3, 50mM KCl,
Performed on every lot of enzyme:
specific PCR on genomic templates with non-optimized primer pairs, activity
check, absence of ribonucleases, absence of nickases and endonucleases,
absence of 3' exonucleases, purity on SDS-PAGE, absence of proteases.
Store at -20°C
SurePRIMEDNA Polymerase is provided with 10 x C buffer (without MgCl2) and with
a separate tube of 25mM MgCl2.
Also available in a convenient Kit!
Chemically modified Taq DNA Polymerase
dNTPs in one kit
SurePRIME™ CORE Kits contain all the reagents (presented as
separate items) required for Hot Start PCR: Qbiogene’s
SurePRIME™ DNA polymerase, high purity dNTPs Mix
(>99%) and optimized incubation buffers.
Two types of kits are supplied:
SurePRIME™ CORE Kit 10 (with dNTPs at 10mM each)
||5 x 250 U
||3 x 500µl (15 µmol each dNTP)
SurePRIME™ CORE Kit 25 (with dNTPs at 25mM each)
||5 x 250 U
||3 x 200µl (15 µmol each dNTP)
information for PCR Process