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Water for DNA Microarrays

 
 
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Impact of Water


Use of water in DNA microarrays

  • Water is used during the manufacturing process. Whether the technique selected is photolithography, printing or electrochemistry, rinsing steps are always necessary, requiring significant amount of high purity water. The photolithography and the electrochemistry techniques also require performing chemical reactions to build and grow the oligonucleotide sequences on the solid surface. This step also requires buffers that must be prepared with high purity water. 
     
  • Microarray experiment. In order to perform the DNA microarray experiment, enough DNA or cDNA needs to be available. It is usually obtained via some kind of PCR-based DNA amplification technique (e.g. simple PCR or reverse-transcriptase-PCR), requiring nuclease-free high purity water. Once the nucleic acid material is available, the hybridization step is carried out to bind the target nucleic acid to the probe – a step that is run in buffer. A washing step concludes the experimental procedure to remove unspecific binding on the solid surface and to the nucleic acid strands. 

Water quality parameters

The high purity water selected should comply with the various steps and materials utilized throughout the DNA microarray experiments. The following steps are involved in the process:
  • DNA microarray manufacturing, i.e. the preparation of the slides/solid support
  • Nucleic acid sample preparation that may require nucleic acid extraction and DNA amplification via PCR
  • DNA microarray experiment, including the hybridization step between the sample DNA and the target
  • Detection step, based on fluorescence or chemiluminescence techniques 
The preparation of the DNA microarrays and the experiments conducted with those microarrays share some common water quality requirements.

Nuclease-free water
In order to prevent the sample as well as the microarray itself from degradation by nucleases, water should be nuclease-free. During the preparation of the microarray, the nucleic acid should also be protected from degradation. Nuclease-free water is also recommended during the nucleic acid extraction and the DNA amplification steps.

Nuclease removal is efficiently done using ultrafiltration devices Point-of use ultrafiltration cartridges can be installed at the outlet of water purification systems to provide nuclease-free water on demand.

Organics
Organics could generate issues during the preparation of the microarrays. In the printing processes, the solid surface must be perfectly clean in order to optimize the printing. The presence of organic, as low as 5 ppb, reduces the efficiency of adhesion of the oligonucleotide onto the surface. In other fabrication processes, where the DNA reacts with the surface of the slide, organics that would come from the water could react non-specifically with the chemical sites of the surface, hence reducing the chance of reaction with the oligonucleotide. Organic molecules could create interferences in the hybridization process.

Water quality has an impact on the detection step (be it by fluorescence or chemiluminescence). Some organics can generate or quench fluorescence, and interfere with the detection of the fluorescent signal, so a low level of organics also optimizes the detection step.

Ions
The concentration of ions is important during the hybridization step. In order to carry out the hybridization in optimum conditions, specific ion concentrations should be maintained. Starting with water virtually free of ions (resistivity 18.2 MΩ•cm) ensures the preparation of the buffers at the right ionic strength and pH.

In addition, polymerase used for PCR needs specific magnesium concentration for maximum efficiency, and is susceptible to inhibition with metals such as cadmium and iron. Therefore, metal-free water should be selected for the PCR-based DNA amplification. For more information on the impact of water contaminants in PCR, please refer to the section on “Water for PCR”.

Silica
Silica can create a film on the solid surface, reducing the efficiency of the printing process. Water with low silica level is therefore highly recommended. Different purification technologies, such as reverse osmosis, electrodeionization and ion exchange resins, are usually combined to remove silica from the water.

Ozone
Ozone is known to contribute to the fading of fluorescent signal, particularly Cy5. The levels of ozone vary with the seasons, with high peaks in summer time. Ozone does not come from the water purification process, but it is present in the air and dissolves in water when water is let standing openly to air. Issues due to ozone may be reduced with careful handling of the high purity water used for the microarrays. Specific spray products have been designed as well to protect the slides from fast fading.

Examples illustrating the impact of water quality on DNA microarrays

A study was carried out to compare DNA microarrays that were prepared with different grades of high purity water (Figure 1). In both cases, resistivity was 18.2 MΩ•cm, ensuring low levels of ions. Both waters had bacteria levels below 1 cfu/mL. The level of organics, however, was different. In experiment A, the level of organics was 22 ppb (µg/L), which could have negatively affected one or more steps in the experiment. The haze prevents good reading of the DNA microarray. In experiment B, the TOC level was 3 ppb, resulting in well defined spots and overall good results.

Water for experiment A was prepared using a combination of Reverse osmosis and ion exchange resins. Water for experiment B was prepared using a combination of reverse osmosis, electrodeionization (Elix technology), activated carbon, UV photooxidation and ion exchange resins.

Figure 1. 
Merck:/Freestyle/LW-Lab-Water/applications/DNA-Microarrays/LW-LC-DNA-Microarrays-Impact-460x457.jpg
Figure 1. Comparison of DNA microarrays obtained using two different water qualities. Experiment A: resistivity 18.2 MΩ•cm, bacteria < 1 cfu/mL, TOC 22 ppb. Experiment B: resistivity 18.2 MΩ•cm, bacteria < 1 cfu/mL, TOC 3 ppb.
In summary, high purity water with high resistivity 18.2 MΩ•cm, low TOC (< 5 ppb, and bacteria free (< 0.1 CFU/mL) is suitable and recommended for DNA microarrays.


More Information
Reference
  • Mabic S, Kano I. Impact of purified water quality on molecular biology experiments. Clin. Chem. Lab. Med., 41 (4), 486-491, 2003
You may find more information related to DNA microarrays in the following web sites:
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