Why use polymerases with higher fidelity or processivity?
Many of today’s techniques demand longer amplifications and greater fidelity than standard Taq DNA polymerase can deliver. Applications such as genome analysis, cloning, sequencing, mutation analysis and protein expression, require not just PCR, but Long, High Fidelity PCR. Standard PCR using Taq DNA polymerase is generally limited to amplifications up to 5 kb. This is due in part to Taq DNA polymerase lacking a 3′→5′ exonuclease or "proofreading" activity, which repairs periodic misincorporations. After a misincorporation, the enzyme will either continue to incorporate nucleotides, causing a processive mistake or a terminal event will occur and elongation will be arrested.
Long and Accurate (LA) PCR is achieved by combining a highly processive thermostable polymerase with a second thermostable polymerase that exhibits a 3′→5′ exonuclease. This blending dramatically increases the length of amplification by using the proofreading polymerse to repair terminal misincorporations. This repair allows the polymerase to resume elongating the growing DNA strand.
AccuTaq™ LA and KlenTaq® LA DNA Polymerase Mixes combine a high quality, highly processive polymerase with a small amount of a thermostable proofreading enzyme. The resulting enzymes mixes are capable of amplifying DNA targets from 0.25 to 40 kb, with an increase in fidelity up to 6.5 times greater than standard Taq DNA polymerase.
Preparation Instructions— Reliable amplification of long DNA sequences requires:
1) effective denaturation of DNA template,
2) adequate extension times to produce large products
3) protection of target DNA from damage by depurination.
For best results, optimize the reaction using the following parameters:
Use of REDAccuTaq—Since the red tracer has no effect on the amplification process, a sample can be easily re-amplified as in “nested PCR”. The presence of the dye also has no effect on automated DNA sequencing, ligation, exonucleolytic PCR product digestion, and transformation. Although exceptions may exist, the dye is generally inert in restriction enzyme digestions. If necessary, the dye can be removed from the amplicon by routine purification methods.
The optimal conditions for PCR will depend on the system being utilized. The following protocol serves only as a reference.
1. Add the following reagents to a thin-walled 0.2 mL or 0.5 mL PCR tube:
*Buffer is provided with enzymes (D8045, D4812, D5809 and D1313)
**Generally, this is the amount of complex target DNA (such as human genomic DNA) required per reaction. Less DNA is needed for amplification of a simple target such as lambda DNA.
*** The final PCR reaction volume can be scaled down to 20 µL by proportionally decreasing each component.
2. Setup a second reaction without template DNA to serve as the no template control.
3. Mix gently and briefly centrifuge to collect all components at the bottom of the tube.
4. Add 50 µL of mineral oil to the top of each tube to prevent evaporation (optional, depending on model of thermal cycler).
5. Optimum cycling parameters vary with PCR composition and thermal cycler. It may be necessary to optimize the cycling parameters to achieve maximum product yield and/or quality.
6. Evaluate the amplified DNA by directly loading 8-10μL of PCR reaction to 0.8 –1% agarose gel and subsequent ethidium bromide staining.6
7. Evaluate the amplified DNA by directly loading 8-10μL of PCR reaction to 0.8 –1% agarose gel and subsequent ethidium bromide staining.6
Note: When amplifying fragments less than 20 kb, the extension time can be reduced according to the fragment size. Normally, a one minute extension time will be sufficient for a 1 kb fragment.