Reliable amplification of long DNA sequences requires: 1) effective denaturation of DNA template, 2) adequate extension times to produce large products and 3) protection of target DNA from damage by depurination. Effective denaturation is accomplished by the use of higher temperatures for shorter periods of time or by the use of co-solvents, such as dimethyl sulfoxide. Addition of DMSO in the reaction at a final concentration between 1 to 4% may increase yield and improve reliability of the system with some complex PCR targets. Betaine (0.8-1.3 M) has been reported to improve the amplification of DNA by reducing the formation of secondary structure in GC rich regions.1
A Perkin-Elmer GeneAmp 2400 cycler was used to develop cycling parameters. Other types of thermal cyclers can also be used, but may require further optimization of cycling parameters.
Primers are usually 21 to 34 bases in length and are designed to have a GC content of 45-50%. Optimally, the melting temperatures of the forward and reverse primers should be within 3 °C of each other and the Tm of the primers should be between 65-72 °C.2 Primers should not have any internal base-pairing sequences (i.e., potential hairpins) or any significant length of complementary regions between the two PCR primers. It is sometimes helpful to design primers with a final CC, GG, CG, or GC on the 3-prime end of the primers in order to increase priming efficiency.3
High quality and adequate length of the template are essential for reliable amplification of larger fragments. Extreme care must be taken in the preparation and handling of the DNA target for long PCR. Nicked or damaged DNA can serve as a potential priming site resulting in high background. Avoid freezing, or, alternatively, freeze only once to minimize damage. The condition of the target DNA is critical. Depurination during cycling is minimized by use of buffers with a pH greater than 9.0 at 25 °C.
Optimization of magnesium concentration may be necessary. Generally magnesium concentrations should be between 1 and 5 mM.
Extension temperature should be limited to 68 °C for optimal performance. Temperatures greater than 68 ºC may result in a reduced amount or no product. For targets greater than 20-kb, extension times should be greater than 20 minutes. Primer annealing and product extension can also be combined into one step if primers are designed to have a Tm between 65‑68 °C. The use of auto-extension is advisable to reduce artifacts. Cycle denaturation times should be kept short. For example, the initial DNA denaturation may be accomplished by a 30-second incubation at 96 °C.
The AccuTaq LA 10x Buffer is at a relatively high pH, and magnesium may precipitate as magnesium hydroxide [Mg(OH)2]. Before use, thaw the buffer at room temperature, then vortex to redissolve any precipitated Mg(OH)2. Alternatively, warm the buffer at 37°C for 3-5 minutes, then vortex.
The optimal conditions for the concentration of REDAccuTaq LA DNA Polymerase, template DNA, primers, and MgCl2 will depend on the system being utilized. It may be necessary to determine the optimal conditions for each individual component.
1. Add the following reagents to a thin-walled 200 µl or 500 µL PCR microcentrifuge tube:
*Typically ≥200 ng template DNA is necessary for amplification of more complex genomes.
2. Mix gently and briefly centrifuge to collect all components to the bottom of the tube.
3. Add 50 µL of mineral oil to the top of each tube to prevent evaporation (optional, depending on model of thermal cycler).
4. The amplification parameters should be optimized for individual primers, template, and thermal cycler. Suggested cycling parameters based on in-house amplification of lambda DNA and a 20kb fragment of human β-globin gene cluster:
5. Evaluate the amplified DNA by agarose gel electrophoresis and subsequent ethidium bromide staining.4
a. When amplifying templates 20 kb or greater, a 15second auto-extension is suggested for cycles 16-30. Some thermal cyclers may not have this auto-extension function; increasing the extension time by 1-4 minute increments is recommended.
b. When amplifying fragments less than 20 kb, the extension time can be reduced according to the fragment size. Normally, one minute extension time will be sufficient for 1 kb fragment.
REDAccuTaq is a registered trademark of Sigma-Aldrich Co. LLC
TWEEN is a registered trademark of Croda International PLC
IGEPAL is a registered trademark of Rhodia Operations.
Use of this product is covered by one or more of the following US patents and corresponding patent claims outside the US: 5,789,224, 5,618,711, 6,127,155 and claims outside the US corresponding to expired US Patent No. 5,079,352. The purchase of this product includes a limited, non-transferable immunity from suit under the foregoing patent claims for using only this amount of product for the purchaser’s own internal research. No right under any other patent claim, no right to perform any patented method, and no right to perform commercial services of any kind, including without limitation reporting the results of purchaser's activities for a fee or other commercial consideration, is conveyed expressly, by implication, or by estoppel. This product is for research use only. Diagnostic uses under Roche patents require a separate license from Roche. Further information on purchasing licenses may be obtained by contacting the Director of Licensing, Applied Biosystems, 850 Lincoln Centre Drive, Foster City, California 94404, USA.
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