dNTP Mediated Hot Start PCR Protocol

Section Overview

• End-point PCR Protocols
• Hot Start dNTP Handling
• Use Guidelines
• Equipment
• Appropriate Analysis Equipment
• Supplies
• Method
• Related Products

End-point PCR Protocols

During PCR assay preparation, nonspecific amplification can occur due to binding of PCR primers to nonspecific templates and from formation of primer dimers which result from using other primer molecules as templates. The protocols presented in this section both adopt controlled PCR activation to allow users to minimize nonspecific amplification while increasing target yield and specificity. Two alternative methods are presented to control amplification of the specific product, these are enzyme and dNTP mediated Hot Start PCR.

Hot Start dNTPs are modified with a thermolabile protecting group at the 3’ terminus. The presence of this modification blocks nucleotide incorporation by DNA polymerase until the nucleotide protecting group is removed during a heat activation step. When standard cycling protocols are employed, a 0–10 minute initial denaturation step at 95 °C facilitates activation. In many PCR applications, Hot Start dNTPs directly replace the natural nucleotides. Although using the Hot Start dNTP Mix, containing the modified nucleoside triphosphates of dA, dC, dG and dT is recommended. It has been observed that replacement of just one or two natural nucleotides with Hot Start dNTPs is sufficient to prevent nonspecific amplification ^6,7. Hot Start dNTPs are available as a blended mix or as a set of individual CleanAmp™ dNTPs (DNTPCA2 or DNTPCA10).

This protocol provides directions for performing standard thermal cycling, single and multiplexed target detection (from 2 to 7 targets) and fast thermal cycling end-point PCR.

Hot Start dNTP Handling

Hot Start dNTPs are similar to natural dNTPs and are stable in aqueous buffer at pH 8–10.5 and at -20 °C for at least one year. When stored incorrectly, the major point for degradation of both natural and Hot Start dNTPs is the triphosphate chain. Much like natural dNTPs, extra care should be taken not to expose Hot Start dNTPs to prolonged temperatures above –20 °C. Restrict exposure of the Hot Start dNTP stock solution to less than 24 hours TOTAL at room temperature. Aliquot stocks to avoid more than 20 freeze–thaw cycles.

1. The Hot Start dNTP Mix is provided as a concentrated 2 μM or 10 μM solution of dATP, dCTP, dGTP and dTTP.
2. The dNTP sets are provided as a 10 μmol solution of each individual dNTP. The dNTPs can be diluted into a PCR buffer solution and frozen at -20 °C in smaller aliquots to ensure stability for at least one year.
3. The dNTPs can be stored for up to 15 days at 4 °C as the dNTP stock solution.
4. Hot Start dNTPs should not be stored at room temperature. They should be thawed at room temperature or on ice (not by heating); mixed by vortexing and pulse centrifugation and then stored on ice during PCR set-up or aliquoting manipulations.

Use Guidelines

1. Both native and recombinant Taq DNA polymerases work well in conjunction with hot start dNTPs in all applications tested. Hot Start dNTPs are also shown to successfully block extension by mesophilic enzymes, such as Klenow DNA polymerase.
2. PCR buffers with a pH range from 8–9 can be used for PCR setup (observed during extensive evaluation by research and development team).
3. For standard thermal cycling protocols, 2.5 mM MgCl_2, 400 μM Hot Start dNTPs and 1.25 units of Taq DNA polymerase is recommended. For improved performance, the Hot Start dNTP concentration can be increased up to 800μM. For every additional 0.2 mM concentration of Hot Start dNTPs, add at least an additional 1.0 mM of MgCl₂ (observed during extensive evaluation by research and development team; see Reverse Transcription).
4. Hot Start dNTPs have been validated for production of amplicons up to 2 kb in length (see Reverse Transcription).
5. When using cDNA as the template, purify the product using a commercially-available clean-up kit to remove unincorporated nucleotides. When using cDNA without purification, this should be no more than 1/10th of the reaction volume of the PCR.
6. In multiplex reactions where four or more targets are to be amplified, the addition of KCl up to 100 mM final concentration will improve results (see Reverse Transcription).

Equipment

• Pipettes dispensing volumes from <1 to 200 μL
  
• Benchtop microcentrifuge
  
• Thermal cycler

Appropriate Analysis Equipment

• Electrophoresis equipment
  
• UV transilluminator
  
• Alternative PCR product analysis system

Supplies

• Sterile filter pipette tips
  
• Sterile 1.5 mL screw-top microcentrifuge tubes (CLS430909)
  
• PCR tubes and plates, select one of the following to match desired format:
  • Individual thin-walled 200 μL PCR tubes (Z374873)
  • Strip tubes, 200 μL (Z374962)
  • 96-well plates (Z374903)
  • 384-well plates (Z374911)
• CleanAmp™ dNTPs (DNTPCA2 or DNTPCA10). Enzyme and compatible buffer, for example one of the following:
  • D4545 which is supplied with a 10× PCR buffer and separate MgCl₂
  • D1806 which is supplied with a 10× PCR buffer containing MgCl₂

Method

1. With the exception of the Hot Start dNTPs and DNA polymerase, thaw the reaction components, vortex to mix, centrifuge briefly and store on ice.
2. Prepare Hot Start dNTPs:
   1. Thaw at room temperature or on ice.
   2. Vortex and pulse centrifuge to thoroughly mix.
   3. If necessary, remove an aliquot of the stock solution and dilute with water or buffer (pH 8–10.5) to desired working concentration.
3. Prepare a master mix containing all components except for the DNA template sample. Add each of the components as shown in Tables 1, 2 or 3 (select appropriate experiment and cycling conditions; multiply amounts by the number of reactions needed) in a microcentrifuge tube, on ice.

Table 1. Reaction Master Mix Components for Hot Start dNTPs Using Standard Cycling Conditions.

Component	Final Concentration	Volume (μL) per Single 25 μL Reaction
10× PCR Buffer without MgCl2	1×	2.5
MgCl2 (25 mM)	2.5 mM	2.5
Forward Primer (10 μM)	20–500 nM	0.04 to 0.1
Reverse Primer (10 μM)	20–500 nM	0.04 to 0.1
Hot Start dNTP solution	0.4 mM	1
Taq DNA polymerase 5 units/μL	0.05 units/μL	0.25
PCR grade water		Up to 20 μL

Table 2. Reaction Master Mix Components for Hot Start dNTPs Using Fast Cycling Conditions.

Component	Final Concentration	Volume (μL) per Single 25 μL Reaction
10× PCR Buffer without MgCl2	1×	2.5
MgCl2 (25 mM)	4.0 mM	4
Forward Primer (10 μM)	20–500 nM	0.04 to 0.1
Reverse Primer (10 μM)	20–500 nM	0.04 to 0.1
Hot Start dNTP solution	0.4 mM	1
Taq DNA polymerase 5 units/μL	0.20 units/μL	1
PCR grade water		Up to 20 μL

Table 3. Reaction Master Mix Components for Hot Start dNTPs for Muliplexed Reactions.

Component	Final Concentration	Volume (μL) per Single 25 μL Reaction
10× PCR Buffer without MgCl2	1×	2.5
MgCl2 (25 mM)	2.5 mM	4
Forward Primer (10 μM)	20–500 nM	0.04 to 0.1
Reverse Primer (10 μM)	20–500 nM	0.04 to 0.1
Hot Start dNTP solution	0.4 mM	1
Taq DNA polymerase 5 units/μL	0.05–0.10 units/ μL	0.25–0.5
PCR grade water		Up to 20 μL

Note: D4545 includes Taq DNA polymerase, 10× PCR buffer (P2317) and 25 mM MgCl₂.

4. Mix the master mix gently to protect the enzyme by pipetting up and down (do not vortex). Pulse spin if necessary.
5. Aliquot 20 μL of reaction master mix into each thin-walled PCR tube.
6. Add 5 μL of the appropriate template DNA to each 20 μL aliquot of master mix for a final reaction volume of 25 μL.
7. Cap, label and pulse spin PCR tubes. Collect reaction solution at bottom of tube.
8. Place the tubes into a thermal cycler with a heated lid and perform the appropriate cycling conditions (Tables 4 and 5).

Table 4. PCR Cycling Conditions for Use with Hot Start dNTPs Under Standard Cycling Conditions and for Multiplexing.

Hot Start dNTP Standard Cycling Conditions	Temp (°C)	Time
Initial Hot Start/denaturation	95	5 min
Steps 1–3 are repeated through 30–40 cycles
Step 1	95	10 sec
Step 2	48–60	1 to 30 sec
Step 3	72	30 sec to 2 min*
Final elongation	72	10 min

*Note: Dependent upon amplicon size: 30 sec for up to 500 bp. Add 1 min for each additional 1 kb.

Table 5. PCR Cycling Conditions for Use with Hot Start dNTPs Under Fast Cycling Conditions.

Hot Start dNTP Fast Cycling Conditions	Temp (°C)	Time
Initial Hot Start/denaturation	98	5 min
Steps 1–2 are repeated through 30–40 cycles
Step 1	95	5 sec
Step 2	65	5 sec

9. Analyze a 10 μL aliquot of the completed reaction by agarose gel electrophoresis or alternative PCR analysis system.

References

1. Kitchen RR, Kubista M, Tichopad A. 2010. Statistical aspects of quantitative real-time PCR experiment design. Methods. 50(4):231-236. https://doi.org/10.1016/j.ymeth.2010.01.025

2. Tichopad A, Kitchen R, Riedmaier I, Becker C, Sta?hlberg A, Kubista M. 2009. Design and Optimization of Reverse-Transcription Quantitative PCR Experiments. 55(10):1816-1823. https://doi.org/10.1373/clinchem.2009.126201

3. Manly B. 1998. Randomization, Bootstrap and Monte Carlo Methods. Methods in Biology. 2nd ed. Chapman Hall:

4. Francis A. 2000. Glove me Tender. The Scientist. [Internet].[updated 14 May 2000]. Available from: https://www.the-scientist.com/technology-profile/glove-me-tender-56007

5. Kellogg D, Rybalkin I, Chen S, Mukhamedova N, Vlasik T, Siebert P, Chenchik A. 1994. TaqStart Antibody:"" hot start"" PCR facilitated by a neutralizing monoclonal antibody directed against Taq DNA polymerase. Biotechniques.. 16(6):1134-7.

6. Koukhareva I, Lebedev A. 2009. 3?-Protected 2?-Deoxynucleoside 5?-Triphosphates as a Tool for Heat-Triggered Activation of Polymerase Chain Reaction. Anal. Chem.. 81(12):4955-4962. https://doi.org/10.1021/ac8026977

7. Koukhareva I, Haoqiang H, Yee J, Shum J, Paul N, Hogrefe RI, Lebedev AV. 2008. Heat Activatable 3'-modified dNTPs: Synthesis and Application for Hot Start PCR. Nucleic Acids Symposium Series. 52(1):259-260. https://doi.org/10.1093/nass/nrn131

8. Tania N, Stephen B. 2013. PCR Technologies: Current Innovations. . 3. CRC Press.

9. Nolan T, Hands RE, Bustin SA. 2006. Quantification of mRNA using real-time RT-PCR. Nat Protoc. 1(3):1559-1582. https://doi.org/10.1038/nprot.2006.236