PCR techniques are in widespread use for the amplification of genetic material. The need for reagents and solutions free from nucleases (DNase, RNase) is widely recognized, however, it is vital to also ensure the absence of other waterborne contaminants which could cause problems with test results.
PCR-based methods, such as reverse transcription PCR (RT-PCR) and quantitative PCR (qPCR), are essential in medical and biological research. DNA amplification by PCR uses DNA polymerase enzymes to synthesize double-stranded DNA molecules from single-stranded ‘templates’, using oligonucleotide primer sequences to target the gene of interest. Both the target specificity and efficiency of the enzyme-catalyzed reaction are highly dependent on the composition of the reaction mixture1. The presence of nucleases – enzymes which cleave the phosphodiester linkages between nucleic acid subunits – will lead to severe disruption of the PCR process, as genetic material will fragment under reaction conditions. It is vital to ensure that all reagents and solutions used in PCR applications are ‘nuclease-free’; however, a number of other contaminants commonly found in water can also impede DNA amplification2, including:
Many bacteria release nucleases and other molecules which interfere with DNA polymerization. The presence of bacterial DNA can lead to errors in qPCR, and amplification of non-target sequences.
The concentration of Mg2+, which is a co-factor for effective substrate binding, is crucial for optimization of polymerase activity3. Other divalent cations interfere with co-factor co-ordination and disrupt substrate binding and trace amounts of heavy metal ions will inhibit enzyme activity.
Negatively charged bio-molecules can reduce substrate turnover by steric interference with substrate binding at the positively charged active sites.
Purifying water for PCR
PCR requires the use of water free from nucleases, micro-organisms, organic compounds and trace elements for all reagents and buffers. Ultrapure grade (Type I) water – with 18.2 MΩ cm resistivity, < 20 ppb total organic carbon (TOC),
ELGA’s PURELAB Ultra Genetic uses UV photo-oxidation, high capacity purification cartridges, combined with an ultrafilter, to reliably deliver ultrapure water for PCR work. Real time resistivity and TOC monitoring ensure verifiable removal of inorganic and organic contaminants and the system offers validated traceability.
Ultrapure water with a high resistivity (18.2 MΩ cm) and free from nucleases, organic compounds and endotoxins should be used for all PCR applications, to ensure optimized amplification of target sequences. To find out more about ELGA LabWater’s water treatment technologies and solutions for life science applications, visit www.elgalabwater.com
- Bogetto, P et al. (2000). Helpful tips for PCR. Focus, 22 (1), 12
- Sambrook, J and Russel, DW (2001). Chapter 8: In vitro Amplification of DNA by the Polymerase Chain Reaction, Molecular Cloning: A Laboratory Manual (3rd ed)
- Yang, L et al. (2004). Critical role of magnesium ions in DNA polymerase beta’s closing and active site assembly, J Am Chem Soc, 126 (27), 8441-8453
- ASTM “ Standard Guide for Bio-applications Grade Water”, D 5196-06.