Method for PCR amplification of Bacterial DNA with low contamination using MTP™ Taq DNA Polymerase

• Background
  
• Reagents
• Procedure
• Materials

Background

MTP™ Taq DNA Polymerase is a recombinant thermostable enzyme from Thermus aquaticus expressed in E. coli and purified using a proprietary process to minimize levels of contaminating DNA. The enzyme has both 5'→3' DNA polymerase and exonuclease activities, is ~95 kDa by SDS-PAGE, and has no detectable endonuclease or 3'→5' exonuclease activities. Each lot of MTP Taq undergoes strict quality control testing to ensure the absence of detectable levels of contaminating DNA.

Contaminating DNA present in most other polymerase preparations often preclude or obscure the accurate interpretation of results, especially when targeting conserved sequences, e.g., bacterial 16S rRNA region.¹ With our proprietary DNA removal methods and strict quality control standards, we can ensure the absence of the most commonly found contaminant DNA. Each lot of MTP Taq is assayed using PCR and primers specific to (1) the conserved region of bacterial 16S rRNA, (2) the Taq expression vector, and (3) the human β-actin gene.

While MTP Taq ensures a high-quality, low contaminant DNA polymerase for reliable PCR amplification, DNA contaminants can be introduced into PCR through a number of other reagents.² To further minimize the risk of contaminant DNA during PCR, we include 10x MTP Taq Buffer with each tube of MTP Taq DNA Polymerase. Each lot of 10x MTP Taq Buffer undergoes the same strict quality control testing as MTP Taq DNA polymerase to ensure the absence of contaminating DNA. To prevent false positive PCR results, only DNA-free reagents should be used in PCR reactions with MTP Taq DNA polymerase.

Figure 1.

Representative QC gel assaying for the presence of conserved 16S rRNA sequence. PCR products run in lanes 1-3 are from "no-template" reactions; PCR products in lanes 4-5, 6-7, and 8-9 are from reactions with 37 fg, 370 fg, and 3.7 pg of E. coli genomic DNA added, Catalog Number D4889.

Unit definition: One unit incorporates 10 nmol of total deoxyribonucleoside triphosphates into acid precipitable DNA in 30 minutes at 74 °C.

Reagents

• MTP™ Taq DNA Polymerase (D7442):
  • MTP Taq DNA Polymerase (D7067)
  • 10X MTP Taq Buffer (M9943)
• PCR grade water (W1754)
• Primers diluted to working concentration (10µM working stocks are sufficient for most assays)
  • Order Custom Oligos here
• DNA to be amplified
• Dedicated pipettes
• Thermal cycler
• Sterile filter pipette tips
• Sterile 1.5 mL screw-top microcentrifuge tubes (such as CLS430909)
• PCR tubes, select one of the following to match desired format:
  • Individual thin-walled 200 µL PCR tubes (Z374873 or P3114)
  • Strip tubes, 200uL (Z374962)
  • Plates
    • 96 well plates (Z374903)
    • 384 well plates (Z374911)
    • Plate seals
      • AlumaSeal® 96 film (Z721549)
• dNTP mix, 10 mM each of dATP, dCTP, dGTP, and dTTP (D7295)
• Thermal cycler
  

Procedure

Note: Every precaution should be taken to avoid contamination of reagents with unknown/unwanted DNA. This includes the following:

• Use a "clean area" for the setup of PCR reactions.² A clean area is a separate lab space (such as a hood) preferably in a separate lab, both of which are free from PCR products. The clean area should contain dedicated lab coats and pipettes (see below) and should be cleansed (either with 10% bleach or UV lamp) after each use. PCR products should never enter a clean area.
• Use sterile, aerosol barrier pipette tips to minimize the risk of aerosol contamination. Change tips after each single use.
• Change gloves frequently, especially after handling DNA.

Amplification Procedure

The optimal conditions for the concentration of MTP 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. It is recommended that the enzyme and the MgCl₂ be titrated to determine the optimal efficiency if the below protocol is shown to be less than satisfactory.

1. Add the following reagents to a suitable PCR tube/plate in the following order:
   

Volume	Reagent	Final Concentration
w µL	Water	-
5 µL	10x MTP Taq buffer	1x
1 µL	10 mM dNTP mix	200 µM each dNTP
x µL	Forward Primer (typically 15-30 bases in length)	0.1–0.5 µM
y µL	Reverse Primer (typically 15-30 bases in length)	0.1–0.5 µM
0.5 µL	MTP Taq DNA Polymerase	0.05 units/µL
z µL	Template DNA (typically 10 ng)	200 pg/µL
50 µL*	Total reaction volume

1. *Maintain final concentrations when scaling reaction volumes. Preparing enzyme / nucleotide / buffer master mixes either with or without primers or template facilitates the setup of multiple reactions.
    
2. Vortex gently to mix. Briefly centrifuge to collect reaction on the bottom of the tube.
    
3. If using a thermal cycler without a heated lid, add 50 µL of mineral oil to the top of each tube to prevent evaporation.
    
   
4. Cycle using thermocycler of choice. The amplification parameters may require optimization for individual primers, templates, and thermal cyclers.
   

Common cycling parameters are:

Step	Temperature	Time	Cycles
Denature	94 ºC	1 minute	1
Denature	94 ºC	1 minute	30
Anneal	5 ºC below primer Tm	15 - 60 seconds
Extend	72 ºC	30 - 65 seconds/kb
Cool	4 ºC	≥ 0 second	1

5. Amplified DNA can be evaluated by standard methods (e.g. agarose gel electrophoresis).³
    
6. If used, mineral oil may be removed by a single 50 µL chloroform extraction.

References

1. Corless CE, Guiver M, Borrow R, Edwards-Jones V, Kaczmarski EB, Fox AJ. 2000. Contamination and Sensitivity Issues with a Real-Time Universal 16S rRNA PCR. 38(5):1747-1752. https://doi.org/10.1128/jcm.38.5.1747-1752.2000

2. Millar BC, Xu J, Moore JE. 2002. Risk Assessment Models and Contamination Management: Implications for Broad-Range Ribosomal DNA PCR as a Diagnostic Tool in Medical Bacteriology. Journal of Clinical Microbiology. 40(5):1575-1580. https://doi.org/10.1128/jcm.40.5.1575-1580.2002

3. Sambrook J, Russel DW. 2000. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press. 3

4. 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

5. Newton CR. 1995. PCR: Essential Data.

6. Choi J, Kim J, Joe C, Kim S, Ha K, Park Y. 1999. Improved cycle sequencing of GC-rich DNA template. Exp Mol Med. 31(1):20-24. https://doi.org/10.1038/emm.1999.3

7. Corless CE, Guiver M, Borrow R, Edwards-Jones V, Kaczmarski EB, Fox AJ. 2000. Contamination and Sensitivity Issues with a Real-Time Universal 16S rRNA PCR. 38(5):1747-1752. https://doi.org/10.1128/jcm.38.5.1747-1752.2000

8. 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

9. Millar BC, Xu J, Moore JE. 2002. Risk Assessment Models and Contamination Management: Implications for Broad-Range Ribosomal DNA PCR as a Diagnostic Tool in Medical Bacteriology. Journal of Clinical Microbiology. 40(5):1575-1580. https://doi.org/10.1128/jcm.40.5.1575-1580.2002

10. 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

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