For hot start PCR
- Chemically modified Taq DNA polymerase
- Low background
- Highly specific amplification
- Ideal for multiplex PCR
SurePRIME™ DNA Polymerase is a new thermostable polymerase suitable for all PCR applications requiring "hot start" conditions. 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.
SurePRIME DNA Polymerase is heat-activated after a pre-incubation step of 15 min at 95°C after which it is functionally equivalent to classical Taq DNA Polymerase. Linear activation is also possible (additional 2 min at 95°C for the first 7-8 PCR cycles). In its inactive state, SurePRIME DNA Polymerase is incapable of extending primer-dimers or mis-annealed primer-template species. Therefore PCR performed after activation is highly specific. In laboratory tests that deliberately accentuate problems commonly encountered in PCR (i.e. nonspecific hybridization, primer-dimer formation) SurePRIME DNA Polymerase easily outperforms Taq DNA polymerase (See Figure 1).
Applications for SurePRIME™ Taq polymerase include:
- Hot start PCR
- Advance preparation of Master Mixes
- Multiplex PCR
- Automated PCR
- Amplification of templates that are GC rich, or contain secondary structure
PCR assays carried out using a non-optimized primer pair, amplifying a well-characterized 400 bp fragment on a human β-globin gene.
Primers are shortened oligos, with additional non-matched bases at the 5' end, designed to create a restriction site at each extremity of the PCR fragment. SurePRIME DNA Polymerase (lane 1: 1 U; lane 2: 2 U; lane 3: 5 U) and classical Taq DNA Polymerase (lane 4: 1 U; lane 5: 2 U; lane 6: 5 U) were comparatively tested. After reaction set-up, the tubes were incubated at 25°C for 30 min to favour misannealing of primers.
For SurePRIME DNA Polymerase: (15 min at 95°C) x 1 - (1 min at 53°C, 2' at 70°C, 1 min at 93°C) x 3 - (1 min at 65°C, 2 min at 70°C, 1 min at 93°C) x 30 - (1 min at 65°C, 10 min at 70°C) x 1
For Taq DNA polymerase: as above omitting the preincubation (15 min at 95°C). Molecular weight marker (MW) is pBR322 Hae III/Taq I.
|Storage Buffer:||20 mM Tris HCl, pH 8.0, 100 mM KCl, 0.1 mM EDTA, 1mM Dithioreithol, 0.5% Tween 20, 0.5% Nonidet P40, 50% Glycerol SurePrime DNA Polymerase is provided with additional storage buffer (200 µl) to be used as dilution buffer.|
|Incubation buffer 1xC:||10 mM Tris HCl, pH 8.3; 50 mM KCl SurePRIME DNA Polymerase 10xC is delivered without MgCl2,provided separately at 25 mM.|
|Quality Control:||Activity, absence of nickases and endonucleases, absence of ribonucleases, specific PCR on genomic template with non-otimized primer pairs.|
|Unit Definition:||One unit is the amount of enzyme required to catalyze the incorporation of 10 nmol of nucleosides into acid-insoluble material in 30 min at 72°C under assay conditions.|