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Long and Accurate PCR Amplification of DNA (D8045)

Preparation Instructions

Reaction Optimization

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 of 1-4% 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

Thermal Cycler
A Perkin-Elmer DNA Cycler 480 has been used to develop cycling parameters. Other types of thermal cyclers can also be used, but may require further optimization of cycling parameters.

Primer design
Primers are usually 21 to 34 bases in length and are designed to have a GC content of 50%. Optimally, the melting temperatures (Tm) of the forward and reverse primers should be within 3 °C of each other and between 60-72 °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

Template
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.

Magnesium concentration
Optimization of magnesium concentration may be necessary. Generally magnesium concentrations should be between 1-5 mM.

Cycle Conditions
Extension temperature should be limited to 68 °C for optimal performance. Temperatures greater than 68 °C may result in a reduced amount of, 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.

Buffer preparation
AccuTaq LA 10X Buffer is at a relatively high pH, and magnesium may precipitate as 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.

Procedures

A. Amplification Procedure for Genomic DNA
The optimal conditions for the concentration of AccuTaq 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:

Volume Reagent Final Concentration
5 µL AccuTaq LA 10x Buffer 1x
2.5 µL dNTP Mix (10 mM each) 500 µM
5 µL Template DNA* (40 ng/uL) 4 ng/µL
1 µL DMSO 2%
1 µL Forward Primer (20pmole/µL) 400 nM
1 µL Reverse Primer (20pmole/µL) 400 nM
34 µL Water, PCR Reagent
0.5 µL AccuTaq LA DNA Polymerase 0.05 units/µL
50 µL Total Volume

*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 λ-globin gene cluster fragments from human genomic DNA):

Initial denaturation 98 °C 30 sec.
For cycles 1-30    
Denaturation 94 °C 5-15 sec.
Annealing 65 °C 20 sec.a
Extension 68 °C 20 min.b
Final extension 68 °C 10 min.


Notes:

a. Oligonucleotides were between 21 bases (high G+C content) and 34 bases (high A+T content) in length. Melting temperatures of oligonucleotides used for amplification of genomic DNA were 62-70°C. This was determined using the algorithm based upon nearest neighbor analysis of Rychlik and Rhoads.2
b.When amplifying templates 20 kb or greater, a 15 second auto-extension is suggested for cycles 16-30. Some thermal cyclers may not have this auto extension function. If it is not available, increasing the extension time by 1-4minute increments is recommended.

5. Evaluate the amplified DNA by agarose gel electrophoresis and subsequent ethidium bromide staining.4

B. Amplification Procedure for Lambda DNA.
The optimal conditions for the concentration of Taq 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:

Volume Reagent Final Concentration
5 µL AccuTaq LA 10x Buffer 1x
2.5 µL dNTP Mix (10 mM each) 500 µM
3 µL Template DNA (2.5 ng/µL) 0.15 ng/µL
3 µL Forward Primer (10 pmole/µL) 600 nM
3 µL Reverse Primer (10 pmole/µL) 600 nM
33 µL Water
0.5 µL AccuTaq LA DNA Polymerase Mix 0.05 units/µL
50 µL Total Volume


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:

Initial denaturation 98 °C 30 sec
For cycles 1-30    
Denaturation 94 °C 5-15 sec
Annealing 65 °C 20 secc
Extension 68 °C 20 mind
Final extension 68 °C 10 min


Notes
:
c. Oligonucleotides were between 21 bases (high G+C content) and 34 bases (high A+T content) in length. Melting temperatures of oligonucleotides used for amplification of genomic DNA were 62-70°C. This was determined using the algorithm based upon nearest neighbor analysis of Rychlik and Rhoads.2
d. When amplifying templates 20 kb or greater, a 15 second auto-extension is suggested for cycles 16-30. Some thermal cyclers may not have this auto-extension function. If it not available, increasing the extension time by 1-4 minute increments is recommended.

5. Evaluate the amplified DNA by agarose gel electrophoresis and subsequent ethidium bromide staining.4

Troubleshooting Guide

No PCR product is observed A PCR component may be missing or degraded. A positive control should always be run to insure components are functioning. A checklist is also recommended when assembling reactions.
There may be too few cycles performed. Increase the number of cycles (3-5 additional cycles at a time).
The annealing temperature may be too high. Decrease the annealing temperature in 2-4 °C increments.
The primers may not be designed optimally. Confirm the accuracy of the sequence information. If the primers are less than 27 nucleotides long, try to lengthen the primer to 27-33 nucleotides. If the primer has a GC content of less than 45%, try to redesign the primer with a GC content of 45-60%.
There may not be enough template. After increasing the number of cycles has shown no success, repeat the reaction with a higher concentration of template.
The template may be of poor quality. Evaluate the template integrity by agarose gel electrophoresis. It may be necessary to repurify template using methods that minimize shearing and nicking.
The denaturation temperature may be too high or too low. Optimize the denaturation temperature by increasing or decreasing the temperature in 1 °C increments.
The denaturation time may be too long or too short. Optimize the denaturation time by increasing or decreasing it in 10 second increments.
The extension time may be tooshort. Increase the extension time in 2-minute increments, especially for long templates.
The reaction may not have enough enzyme. 0.5 µL (2.5 units) is sufficient for most applications. It is recommended that the cycling parameters be optimized before the enzyme concentration is increased. In rare cases, the yields can be improved by increasing the enzyme concentration. However, if the enzyme amount is above 1 µL (5 units), higher background levels may be seen
Mg++ levels may be too low. This is unlikely if the 10x reaction buffer (provided) is used and the deoxynucleotides do not exceed a concentration of 0.6 mM each. However, if the concentration of EDTA in the sample is greater than 5 mM, this can reduce the effective concentration of magnesium.
Deoxynucleotides are too low. This is unlikely if the final concentration of each deoxynucleotide is 0.5 mM. This concentration of dNTPs is suitable for a wide range of applications. If the dNTPs are being prepared in the laboratory, be sure that the final concentration of each deoxynucleotide is 0.5 mM. If the concentration of dNTPs is increased, the Mg++ concentration will need to be increased proportionately.
Target template is difficult. In most cases, inherently difficult targets are due to unusually high GC content and /or secondary structure. Betaine has been reported to help amplification of high GC content templates at a concentration of 0.8-1.3 M. In some cases, the addition of 1-4% DMSO may help.
Multiple products There may be too many cycles performed. By reducing the cycle number, the nonspecific bands may be eliminated.
The annealing temperature may be too low. Increase the annealing/extension temperature in increments of 2-3 °C.
The primers may not be designed optimally. Confirm the accuracy of the sequence information. If the primers are less than 27 nucleotides long, try to lengthen the primers to 27-33 nucleotides. If the primer has a GC content of less than 45%, try to redesign the primers with a GC content of 45-60%.
Touchdown PCR may be needed. “Touchdown” PCR significantly improves the specificity of many PCR reactions in various applications. Touchdown PCR involves using an annealing/extension temperature that is higher than the Tm of the primers during the initial PCR cycles. The annealing/extension temperature is then reduced to the primer Tm for the remaining PCR cycles. The change can be performed in a single step or in increments over several cycles.5
Products are smeared Too many cycles may have been performed. Reduce the cycle number in 3-5 cycle increments.
The denaturation temperature may be too low. Increase the denaturation temperature in 1 °C increments.
The extension time may be too long. Decrease the extension time in 1-2 minute increments
Touchdown PCR may be needed. See recommendations under “Multiple Products” for procedure.
There may be too much enzyme in the reaction mix. 0.5 µL (2.5 units) is sufficient for most applications. However, this concentration may be too high for some applications. It is recommended the cycling parameters be optimized first, as described above, then, reduce the enzyme concentration to 0.5‑0.2X.
Magnesium concentration may be too high. The magnesium concentration has been optimized in the supplied 10x reaction buffer. If the concentration of the dNTPs is 0.5 mM, it is very unlikely that the magnesium concentration is too high.
The template concentration may be too high. Reduce the concentration of the template in the PCR reaction.
Product is wrong size The primers may not be designed optimally. See recommendations under “Multiple Products” for procedure.
The extension time may be too short. Increase the extension times in 2-minute increments or use Touchdown PCR.
Faint Product There may not be enough template. Add additional template in 50 ng increments for genomic or 1‑2 ng for viral.
There may be too few cycles performed. Increase the cycle number in 3-5 cycle increments
The extension time may be too short. Increase the extension times in 2 minute increments
A co-solvent may be required Add DMSO (1-4%), or 0.8-1.3 M betaine, final concentration

Materials

     

References


  1. Rees, W. A., et al. Betaine can eliminate the base pair composition dependence of DNA melting. Biochemistry, 32, 137-44 (1993).
  2. Rychlik, W., and Rhoads, R. E., A computer program for choosing optimal oligonucleotides for filter hybridization, sequencing and in vitro amplification of DNA. Nucleic Acids Res., 17, 8543-8551 (1989).
  3. Lowe, T., et al., A computer program for selection of oligonucleotide primers for polymerase chain reaction. Nucleic Acids Res. 18, 1757-1761 (1990).
  4. Molecular Cloning: A Laboratory Manual, Third Edition, Sambrook, J., et al.,(Eds.) (Cold Spring Harbor Laboratory Press, New York, 2000).
  5. Don, R. H., et al. “Touchdown” PCR to circumvent spurious priming during gene amplification. Nucleic Acids Res., 19, 4008 (1991).
  6. Barnes, W. M., PCR amplification of up to 35-kb DNA with high fidelity and high yield from l bacteriophage templates. Proc. Natl. Acad. Sci. USA, 91, 2216-2220 (1994).
  7. Cheng, S., et al., Effective amplification of long targets from cloned inserts and human genomic DNA. Proc. Natl. Acad. Sci. USA, 91, 5695-5699 (1994).
  8. Frey, B., and Suppmann, B., Demonstration of the Expand™ PCR system’s greater fidelity and higher yields with a lacl-based PCR fidelity assay. Biochemica, 2, 8-9 (1995).
  9. PCR Technology: Current Innovations, Griffin, H. G., and Griffin, A. M., (Eds.) (CRC Press, Boca Raton, FL, 1994). Catalog No. Z357499
  10. PCR Strategies, Innis, M. A., et al. (Eds.) (Academic Press, New York, 1995). Catalog No.  Z364452
  11. PCR Protocols: A Guide to Methods and Applications, Innis, M., et al. (Eds.) (Academic Press, San Diego, California, 1990). Catalog No.  P8177
  12. Nelson, et al. The fidelity of TaqPlus™ DNA Polymerase in PCR. Strategies Mol. Biol., 8, 24-25 (1995).
  13. PCR: Essential Data Series, Newton, C. R., (Ed.) (John Wiley & Sons, New York, 1995). Catalog No. Z364916.
  14. Roux, K.H. Optimization and troubleshooting in PCR. PCR Methods Appl., 4, 5185-5194 (1995).

 

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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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