End Point PCR Protocol for Long and Accurate DNA Amplification

Technology Overview
Equipment
Reagents
Assay Considerations
Procedure
Troubleshooting
Materials
References

Technology Overview

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.

Equipment

• pipettes dispensing volumes from <1 to 200 μL
• benchtop microcentrifuge
• thermal cycler
• electrophoresis equipment
• UV transilluminator

Reagents

• Enzyme and buffer, review the following table to define optimal reagents for your application:

• Sterile filter pipette tips
  
• PCR tubes, select one of the following to match desired format:
  • Individual thin-walled 200 µL PCR tubes (Z374873 or P3114)
  • Individual thin-walled 650 µL PCR tubes (Z374873)
  • strip tubes, 200uL (Z374962)
  • Plates
    • 96 well plates (Z374903)
    • Plate seals
      • AlumaSeal^® 96 film (Z721549)
        
        
• dNTP mix, 10 mM each of dATP, dCTP, dGTP, and dTTP (D7295)
• PCR grade water (W1754)
• DNA template
• Primers diluted to working concentration (10µM working stocks are sufficient for most assays)
  • Order Custom Oligos here
    
• DNA marker, select appropriate marker based upon your PCR amplicon size

Assay Considerations

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:

• Thermal Cycler
  • The Perkin-Elmer DNA Cyclers 480 and 9700 have been used to develop the 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 long and are designed to have a GC content of 45-60%.
  • 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.
  • Primers should not have any internal base-pairing sequences (i.e., potential hairpins) or complementary regions of any significant length between the two PCR primers.
• Template
  • An intact, high quality template is 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.
  • Depurination during cycling is minimized by use of buffers with a pH greater than 9.0 at 25 °C. This higher pH limits potential depurination damage to DNA.
• Magnesium concentration
  • Optimization of magnesium concentration may be necessary. Generally magnesium concentrations should be between 1 and 5 mM.
• Cycling
  • Effective denaturation is accomplished by using higher temperatures for shorter periods of time.
  • The extension temperature should be limited to 68 °C for optimal performance. Temperatures greater than 68 °C may result in reduced 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 equal to or greater than 70 °C.
• Buffer preparation
  • 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.
• Hot-start options
  • Hot- start dNTPs (DNTPCA1) can be used in assay reactions for either D8045 or D4812 to add hot-start

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.

Procedure

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.