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:

Long and Accurate DNA Polymerase
without Hot-Start		with Hot-start
Clear formulation without dye	With red dye for direct load on gels	Clear formulation without dye	With red dye for direct load on gels
AccuTaq™ LA DNA Polymerase (D8045)	REDAccuTaq® LA DNA Polymerase (D4812)	JumpStart™ AccuTaq™ LA DNA Polymerase (D5809)	JumpStart™ REDAccuTaq® LA DNA Polymerase (D1313)

• 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

Product Name (Product Number)	DirectLoad™ 1 kb DNA Ladder (D3937)	DNA Ladder, Supercoiled (D5292)	DirectLoad™ Wide Range DNA Marker (D7058)
Size Range	500bp -10,000bp	2,067bp – 16,210bp	50bp -10,000bp
Picture of ladder	(0.75% gel)	(0.7% gel)	(0.75% gel)

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:

Reagent	Final Concentration	Volume
AccuTaq LA 10 x Buffer (B0174)*	1x	5 µL
dNTP mix (D7295)	500 µM	2.5 µL
Template DNA	200-500 ng total**	- µL
Forward primer (10 µM)	0.4 µM	2 µL
Reverse primer (10 µM)	0.4 µM	2 µL
Water, PCR Reagent (W1754)	----	q.s.
Long and Accurate PCR Enzyme (1 unit/µL)	0.05 units/µL	2.5 µL
Total Volume***		q. 50 µL

*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.

Typical cycling parameters for a 20kb genomic DNA fragment

Initial denaturation	96 °C	30 sec
For cycle 1-30:
Denaturation	94 °C	5-15 sec
Annealing	62-65 °C	30 sec
Extension	68 °C	20 -25 min
Final extension	68 °C	30 min
Hold	4 °C

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

Typical cycling parameters for a 20kb genomic DNA fragment

Initial denaturation	96 °C	30 sec
For cycle 1-30:
Denaturation	94 °C	5-15 sec
Annealing	62-65 °C	30 sec
Extension	68 °C	20 -25 min
Final extension	68 °C	30 min
Hold	4 °C

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.

Troubleshooting Guide

Problem	Possible Cause	Solution
No PCR product is observed	A PCR component is missing or degraded.	A positive control should always be run to insure components are functioning. A checklist is also recommended when assembling reactions.
	Too few cycles were performed.	Increase the number of cycles (3-5 additional cycles at a time).
	The annealing temperature is too high.	Decrease the annealing temperature in 2-4 °C increments.
	The primers are not 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 is not enough template.	After increasing the number of cycles has shown no success, repeat the reaction with a higher concentration of template.
	The template is of poor quality.	Evaluate the template integrity by gel electrophoresis. It may be necessary to repurify template using methods that minimize shearing and nicking.
	The denaturation temperature is too high or too low.	Optimize the denaturation temperature by increasing or decreasing the temperature in 1 °C increments.
	The denaturation time is too long or too short.	Optimize the denaturation time by increasing or decreasing it in 10-second increments.
	The extension time is too short.	Increase the extension time in 2-minute increments, especially for long templates.
No PCR product is observed (continued)	The reaction does not have enough enzyme.	1.0 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 2 µL (5 units), higher background levels may be seen.
	Mg2+ levels are suboptimal.	This is unlikely if the 10X reaction buffer (with MgCl2) is used and the deoxynucleotides do not exceed a concentration of 0.6 mM each (as deoxynucleotide triphosphates can bind Mg2+). Typically, MgCl2 is optimized between 1 to 5 mM. Also, EDTA present in the sample at greater than 5 mM will reduce the effective concentration of magnesium.
	Deoxynucleotide concentration is 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 Mg2+ concentration will need to be increased proportionately.
	Target template is complex.	In most cases, inherently complex targets are due to unusually high GC content and/or secondary structure.
There are multiple or smeared products	The annealing temperature is too low.	Increase the annealing temperature in increments of 2-3 °C.
	The primers are not 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 required.	“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.7
	Too many cycles were performed.	The nonspecific bands may be eliminated by reducing the number of cycles.
	There is too much enzyme in the reaction mix.	1 µL (2.5 units) is sufficient for most applications. However, this concentration may be too high for some applications. We recommend optimizing the cycling parameters first as described above, then if necessary incrementally reduce the enzyme concentration to determine the optimal concentration.
There are multiple or smeared products (continued)	Magnesium concentration is too high.	The MgCl2 concentration should be optimized. Typically, the concentration of MgCl2 is optimal between 1 and 5 mM. If the concentration of the dNTPs is 0.5 mM, it is very unlikely that the magnesium concentration is too high.
	The template concentration is too high.	Reduce the concentration of the template in the PCR reaction.
	The template concentration is too low.	Add additional template in 50 ng increments for genomic DNA or 1-2 ng for viral DNA.
There is no reduction of nonspecific PCR bands when using the JumpStart enzyme.	The antibody affinity may be reduced by reaction components or conditions.	Some cosolvents, solutes (e.g., salts) and pH extremes may reduce the affinity of the JumpStart Taq antibody for the polymerase and thereby compromise its effectiveness. Check your reaction mixture and conditions and/or check your system with a manual hot start method.
	Primers were not designed appropriately.	Check your system with a manual hot start method. If the results are similar, raise the annealing temperature in 2-3 °C increments to improve the specificity of binding. If raising the temperature reduces the yield of the specific product with only a small reduction of side reaction products, it may be necessary to redesign the primers.8
	There was crossover contamination of specific and/or nonspecific PCR products.	Take special precautions to avoid crossover contamination of PCR reactions, including primer-dimer artifacts.9
The yield of specific product is low.	Too few cycles were performed.	Increase the cycle number in 3-5 cycle increments.
	Extension times are too short.	Increase the extension times in 2-minute increments.
	A co-solvent is required.	Add dimethyl sulfoxide up to a final concentration of 5%.
	PCR priming opportunities may be low due to reaction conditions or primer design.	Modify the reaction conditions by increasing the denaturation temperature to 95 °C, increase extension times in 2-minute increments, increase MgCl2 and dNTP concentrations, etc. Redesign PCR primers.

References

1. Barnes W. 1994. Proc. Natl. Acad. Sci. USA.(91):2216-2220.

2. Cheng S, ea. 1994. Proc. Natl. Acad. Sci. USA.(91):5695-5699.

3. Rychlik W, Rhoads RE. 1989. A computer program for choosing optimal oligonudeotides for filter hybridization, sequencing andin vitroamplification of DNA. Nucl Acids Res. 17(21):8543-8551. https://doi.org/10.1093/nar/17.21.8543

4. Lowe T, Sharefkin J, Yang SQ, Dieffenbach CW. 1990. A computer program for selection of oligonucleotide primers for polymerase chain reactions. Nucl Acids Res. 18(7):1757-1761. https://doi.org/10.1093/nar/18.7.1757

5. Roux KH. 1995. Optimization and troubleshooting in PCR.. Genome Research. 4(5):S185-S194. https://doi.org/10.1101/gr.4.5.s185

6. Sambrook J, ea. 2000. Third Edition, Cold Spring Harbor Laboratory Press, New York. Molecular Cloning: A Laboratory Manual.Catalog Number M8265.

7. Rees WA, Yager TD, Korte J, Von Hippel PH. 1993. Betaine can eliminate the base pair composition dependence of DNA melting. Biochemistry. 32(1):137-144. https://doi.org/10.1021/bi00052a019

8. Don R, Cox PT, Wainwright B, Baker K, Mattick JS. 1991. ?Touchdown? PCR to circumvent spurious priming during gene amplification. Nucl Acids Res. 19(14):4008-4008. https://doi.org/10.1093/nar/19.14.4008

9. Huang L, Jeang K. 1994. Biotechniques.(16):242-246 .

10. Kwok S, Higuchi R. 1989. Avoiding false positives with PCR. Nature. 339(6221):237-238. https://doi.org/10.1038/339237a0