Water for DHPLC
Impact of Water
Impact of water quality on DHPLC
In the following experiment, two types of water sources were compared for their relative impact on the DHPLC separations. High purity water freshly produced was compared to several brands on HPLC-grade bottled water.Routinely, PCR and heteroduplex formation from DNA that contains a heterozygous single base pair change will result in the presence of both heteroduplex and homoduplex DNA species. Such base pair changes (mutations) can be identified by detecting heteroduplexes via reversed-phase HPLC performed using DNASepTM (Transgenomic, Omaha,NE) chromatography media in conjunction with the ion pairing reagent, triethyl ammonium acetate (TEAA). TEAA is used to facilitate DNA binding to the DNASep matrix. DNA bound to the matrix is eluted from the column using an increasingly hydrophobic water-acetonitrile gradient. The process selectively resolves DNA molecules by differences in size as well as by differences in helical composition induced by temperature-modulated (heat) denaturation.
Therefore, DNA species of the same size, but with differing denaturation profiles corresponding to base pair changes, can be resolved.
Materials and Methods: Conditions to Verify DNASep Column Resolution
Successful mutation detection requires the use of positive controls to validate results. We have used the Life Technologies (Life Technologies, Rockville, MD) 100 base pair (bp) ladder as a DNA standard to check the performance of the DNASep column. The DNA standard ladder used consists of fragments that differ by 100-bp between 100-bp and 1500-bp. The 600-bp fragment is more intense than the other fragments making it easy to identify. The ladder standard was analyzed using the following conditions:
- LC System: WAVE™ (Transgenomic, Omaha, NE) Fragment Analysis System
- Column: DNASep 35mm x 6.5mm column
- Detection: 260 nm
- Flow rate: 0.9 ml/min
- Buffer A: 0.1M TEAA
- Buffer B: 0.1M TEAA, 25% acetonitrile
- Gradient: 40% to 72% buffer B over 16 minutes
- Temperature: 50 °C
Results: The Influence of Reagent Water Sources
Transgenomic guarantees that the DNAsep column can perform 4000 injections. When analyzing the ladder standard, there was a significant degradation in resolution and decreased number of injection runs when using bottled HPLC-grade water versus Milli-Q® water in the aqueous mobile phase. Poor resolution of DNA species on the column rendered accurate mutation detection impossible.
When using bottled HPLC-grade water for initial runs, a new column successfully resolved the DNA ladder with four peaks resolved between the prominent 600-bp peak and the 1500-bp peak (figure 1). However, column performance deteriorated after 2167 injections as shown by a loss of resolution between the strong 600-bp and 1500-bp fragments and a general loss of peak definition (figure 2). Performance on columns run using bottled water deteriorated after from 408 to 2167 injections resulting in a 50% to 90% loss of expected column life (table 1).
A dramatic improvement in performance was seen by using Milli-Q® water. After over 6000 and 10,000 injections, the DNA ladder standard was resolved with no detectable loss in original column performance (figure 3). The number of injections possible using Milli-Q® water was up to 25 times greater than the number of injections possible using bottled water (table 1).
The organic and ionic purity of reagent waters varied considerably. Analysis via ICP-MS revealed high levels of sodium, potassium and other metal ions in the bottled water sources versus Milli-Q® water (table 2). Additionally, there were high levels of dissolved organic contaminants in the bottled waters versus Milli-Q water (table 3).
It is critical that chemical reagents of high purity, including water, be used during the process. Interference from contaminating organics or metal ions retained on the column were suspected causes of column failure. It is suspected that impurities found in the bottled water degraded the ability of the column to retain and resolve DNA. The glass containers, metal closures, and plastic or rubber seals of the reagent water bottles may leach water-soluble impurities into the bulk water during long periods of storage. Divalent and trivalent metal cations, especially dissolved iron, have been reported to irreversibly bind to DNA and interfere with the resolution of DNA species during DHPLC.
Additionally, organic contaminants have been associated with column fouling in which adsorbed organic materials occlude the binding sites of the chromatography media. Elevated organic levels, measured as Total Organic Carbon, result from either extraction of organic impurities from wetted surfaces or poor performance of the water purification system used to deliver reagent water to the bottles. The result of column fouling is a deterioration of column performance seen in poor retention and loss of resolution.
Recovery of column effectiveness is dependent on the nature and extent of the adsorbed contamination, and increased throughput magnifies the fouling effect.
Conclusion
Use of water newly purified with the Milli-Q® water system significantly improved the performance of the DHPLC method as seen by the increased number of injections. It is critical, however, to minimize the subsequent storage of high-purity water in bottles or carboys after dispensing. Care should be taken to thoroughly rinse reagent reservoirs used in chromatography stations, and reservoirs should be closed to open air and filled with newly prepared water or buffers.
More Information
Literature- Mabic S, Kano I. Impact of purified water quality on molecular biology experiments. Clin. Chem. Lab. Med., 41 (4), 486-491, 2003.
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