Thermolase DNA polymerase used in combination with supplied buffer provides reproducible results in a wide range of PCR applications without the need for optimization. These include amplification from genomic DNA and cDNA templates, amplification of plasmid DNA for subsequent cloning, differential display, DNA fingerprinting, RAPD-PCR etc. The addition of acetamide (to 5%) or T4 gp32 protein (to 1 nM) increases specificity of the amplification reaction. Also longer fragments up to 9 kb can be succesfully amplified from different range of templates.
If PCR sensitivity is low, a nested PCR method can increase PCR product yield. Nested PCR involves two rounds of amplification reactions. The first-round PCR is performed according to hot-start protocol. Subsequently, an aliquot of the first-round PCR product, for example 1 µl of a 1:10 dilution, is subjected to a second round of PCR. The second-round PCR is performed with two new primers that hybridize to sequences internal to the first-round primer-target sequences. In this way, only specific first-round PCR products will be amplified in the second round.
Hot-start protocol provides higher specificity and minimizes background. It is applied for amplification of complex cDNA templates, low-copy targets or in multiplex PCR. There are several methods to reduce the amplification of non-specific targets (mis-priming) and primer-oligomerization that occurs mainly during pre-PCR setup. In manual hot start, one component (usually polymerase or template) is omitted in master mix before thermal cycling is started. After all tubes have been loaded into thermal block of PCR maschine and the temperature has been raised above annealing temperature of the primer (usually 70-80°C), the omitted reagent is added separately to each tube. The separation of the essential component can be accomplished by physical barriers, such as wax plug. During the initial denaturation step, the wax melts and allows all reaction components to mix.
ThermolaseAB is formulated with antibody against the polymerase domain which specifically blocks DNA polymerase activity during setup of PCR reaction. Heat-labile antibody is irreversible inactivated during denaturation step.
ThermolaseTA is a chemically modified DNA polymerase for hot start application. The enzyme is inactive at ambient temperatures and can be irreversible activated at high temperatures (5-10 min at 95°C). Polymerase activity is completely restored by prolonged incubation at elevated temperatures.
Long-template and accurate PCR
Some PCR applications, such as cloning of cDNA sequences for protein expression, sequencing and mutation analysis, need DNA polymerases that have elevated fidelity. ThermolasePR is a single-enzyme system that offers higher fidelity but lower processivity as compared to Thermolase. ThermolaseHFLP is a combination of two enzymes, a higly processive polymerase and a polymerase with proofreading activity, created to amplify longer DNA templates with higher fidelity.
Amplification of GC-rich sequences and templates with strong secondary structures
Compatible solutes, such as ectoin and betain, can reduce the base pair composition dependence of DNA melting. The addition of ectoine or betaine at a final concentration of 1-1,7 M reduces melting temperature of GC-rich DNA up to 6°C and improves the amplification of GC-rich DNA or templates that form strong secondary structures (e.g., repeats). In some cases, the addition of betaine has no effect on amplification or can reduce efficiency of amplification reaction. In this later case, make a titration with 5 M solution of betaine from 0,5 to 2,5 M. Betaine solutions should not be stored for a longer time.
Quantitative real-time PCR with hybridization probes
ThermoKlenAB DNA polymerase lacking an endonucleolytic activity in combination with specific antibody suppressing the formation of nonspecific products is your choice for quantitative real-time PCR with hybridization probes.
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