Antibody-Enzyme Mediated Hot Start PCR Protocol

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.


When using hot start Taq DNA polymerase, the enzyme remains inactive until heated. Hot Start DNA polymerase control is achieved by chemical or antibody modification of the enzyme. Chemically modified hot start enzymes require up to 10 minutes activation whereas antibody mediated hot start enzymes are activated within 1 minute.

JumpStart™ Taq DNA Polymerase (Sigma) is an antibody inactivated, Hot Start enzyme. During the initial denaturation step of the PCR, the antibody is also denatured and dissociates from the DNA Polymerase, therefore enzyme activity is restored. The resulting PCR exhibits a higher specificity and yield5.

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

Resolution of DNA Size Standards Through Agarose Gel

Figure P2-17. Resolution of DNA Size Standards Through Agarose Gel.

Table P2-1. Hot Start DNA Polymerase Enzymes and Buffers (Sigma).

Hot Start DNA Polymerase
Standard Format–Separate Components                 ReadyMix–Premixed PCR Master Mix
Containing MgCl2
Separate MgCl2
(for MgCl2 Optimization)
Containing MgCl2
With red dye for direct
load on gels
Clear formulation
without dye
Clear formulation
without dye
Clear formulation
without dye
With red dye for direct
load on gels
JumpStart™ REDTaq® DNA
Polymerase
Cat. No. D8187
JumpStart™ Taq DNA
Polymerase, with MgCl2
Cat. No. D9307
JumpStart™ Taq DNA
Polymerase, without MgCl2
Cat. No. D4184
JumpStart™ Taq
ReadyMix™
Cat. No. P2893
JumpStart™ REDTaq®
ReadyMix™
Cat. No. P1107

 

  • PCR grade water (Sigma W1754 or dispense Sigma W4502 into 20 mL aliquots and freeze; use a fresh aliquot for each reaction).
  • DNA/cDNA template:
    • cDNA reaction diluted 1:10 to detect medium to highly expressed targets or between 1:2 to 1:5 for rare transcripts.
    • gDNA 10 ng to 100 ng.
  • Primers diluted to working concentration (10 μM working stocks are appropriate for most assays but multiplex blends may require mixing of 100 μM stocks).
    • Custom oligos can be designed according to the OligoArchitect Online (see OligoArchitect Assay Design) and can be ordered at sigma.com/oligos.

Method

1.    Leaving the DNA polymerase on ice or at –20 °C, thaw the remaining reaction components on ice, vortex to mix
       (except the enzyme), centrifuge briefly and replace on ice.

2.    Set up PCR reactions:
       a. Prepare a master mix containing all reaction components with the exception of the DNA/cDNA template
           (Tables P2‑2A or P2‑2B). Calculate the master mix required by multiplying amounts by the number of
           reactions needed, including controls and then add 10% to ensure a sufficient quantity for all samples.

Table P2-2A. Reaction Master Mix Components for Standard Format Hot Start DNA Polymerases.

Component Final Concentration
(in a 25 μL Reaction)
Master Mix Volume (μL)
per Single 25 μL Reaction
PCR buffer (10×) 2.5
Magnesium chloride (50 mM)* 2.5 mM 1.25
Forward/reverse primer
(10 μM stock)
50–500 nM 0.1 to 1
dNTP mixture (10 mM of each
nucleotide)
0.2 mM 0.5
Taq DNA polymerase (5 U/μL) 0.05 units/μL 0.25
PCR grade water Up to 20 μL Up to 20

*Note: Magnesium chloride is added separately if not already in the PCR buffer or when previous optimization has revealed a requirement for a higher concentration.

 

Table P2-2B. Reaction Master Mix Components for ReadyMix Format Hot Start DNA Polymerases.

Component Final Concentration
(in a 25 μL Reaction)
Master Mix Volume (μL)
per Single 25 μL Reaction
JumpStart Taq ReadyMix (2×) 12.5
Forward/reverse primer
(10 μM stock)
50–500 nM 0.1 to 1
PCR grade water Up to 20 μL Up to 20

 

       b. Combine reaction components into a 1.5 mL microcentrifuge tube on ice.

3.    Mix the reaction master mix by carefully pipetting up and down, ensuring that all mix is expelled from the
       pipette tip and then pulse or centrifuge briefly to collect the sample at the bottom of the tube.

4.    Aliquot 20 μL of master mix into the required numer of 200 μL thin-walled PCR tubes (label/number the tubes
       containing samples, including replicates and controls).

5.    Add 5 μL of the DNA template sample (containing a total of 10 ng to 100 ng gDNA or dilute a cDNA sample
       1:2 to 1:10) to reach a final reaction volume of 25 μL.

6.    Spin the PCR tubes and place into a thermal cycler with a heated lid.

7.    Determine the appropriate Ta for the primers. A good first test can be performed using a Ta that is 5 °C lower
       than the Tm of the primer with the lowest Tm.

8.    Determine the required thermal cycling protocol with reference to Table P2-3.

9.    End the PCR with an incubation at 72 °C for 10 min to ensure that all products are full length.

Table P2-3. PCR Cycling Conditions for Use with Antibody Inactivated Hot Start DNA Polymerases.

Hot Start Cycling Conditions Temp (°C) Time
Initial Hot Start/denaturation 95 1 min
Steps 1–3 are repeated through 25–50 cycles
Step 1 95
30 sec
Step 2
48-60 30 sec
Step 3
72
30 sec to 2 min*
PCR Completion 72 10 min

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

 

10.   Analyze a 10 μL aliquot of the completed reaction by agarose gel electrophoresis, with visualization on
        transilluminator or other chosen analysis method.

Materials

     

References

  1. Kitchen, R.R., Kubista, M., Tichopad, A. Statistical aspects of quantitative real-time PCR experiment design. Methods 2010; 50: 231-236.
  2. Tichopad, A., Kitchen, R., Riedmaier, I., et al. Design and optimization of reverse-transcription quantitative PCR experiments. Clin Chem 2009; 55: 1816-1823.
  3. Manly, B. Randomization, Bootstrap and Monte Carlo Methods. Methods in Biology 2nd ed Chapman Hall; 1998.
  4. Francis, A. Glove me Tender. The Scientist 15th May, 2000.
  5. Kellogg, D.E., Rybalkin, I., Chen, S., et al. TaqStart Antibody: “hot start” PCR facilitated by a neutralizing monoclonal antibody directed against Taq DNA polymerase. Biotechniques 1994; 16: 1134-1137.
  6. Koukhareva, I., Lebedev, A. 3’-Protected 2’-deoxynucleoside 5’-triphosphates as a tool for heat-triggered activation of polymerase chain reaction. Anal Chem 2009; 81: 4955-4962.
  7. Koukhareva, I., Haoqiang, H., Yee, J., et al. Heat activatable 3’-modified dNTPs: synthesis and application for hot start PCR. Nucleic Acids Symp Ser (Oxf ) 2008; 259-260.
  8. PCR Technologies: Current Innovations. 3 ed. Edited Tania Nolan and Stephen Bustin CRC Press; 2013.
  9. Nolan, T., Hands, R.E., Bustin, S.A. Quantification of mRNA using real-time RT-PCR. Nat Protoc 2006; 1: 1559-1582.