SurePRIME DNA Polymerase


Chemically modified for use in "Hot Start" PCR

SurePRIME™ 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.

Chemically 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.

Easy to use
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.

Save time and effort
Switching to SurePRIME™ avoids wasting time with complicated and time-consuming manual "hot start" procedures or less efficient antibody-binding solutions

Experiment 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.

To further 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 of reactions.

		SurePRIMEPCR Primers Oligo1 5’ CTG GCC ACT CAC CTT AGG GT 3’ Tm = 36 °C for specific bases (black) 60°C for whole oligo - 20 nts Oligo2 5’ TCG CAG TTC TGG GCA TAA AAG 3’ Tm = 34 °C for specific bases (black) 58 °C for whole oligo - 21 nts PCR Programs Taq Polymerase [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 “Hot start” enzymes [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
Figure 1 Lane 1: 1U Lane 2: 2U Lane 3: 5 U of Taq DNA polymerase, respectively with 100 ng template DNA Lane 4: pBR322/HaeIII	Figure 2 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.

Conclusions

• The enzyme exhibits no activity at room temperature. This means that variations in the time taken to set up reactions will not affect reproducibility of results.
• SurePRIMEDNA polymerase provides highly specific DNA amplification even when using non-optimized primers.Unit definition

One unit 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.

Quality control
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.

Storage: 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!

SurePRIME™ CORE Kits


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)

SurePRIME™ CORE Kit 25 (with dNTPs at 25mM each)

 Patent information for PCR Process

Related products
 SurePRIME-&GO™ Mastermix