Archived | DNA Extraction and Quantitation for Forensic Analysts | DNA Purification

Step Four: DNA Purification

Photo of centrifugal filter unit — National Institute of Justice (NIJ) (see reuse policy).

DNA can be recovered from the aqueous phase with an ethanol precipitation or using a centrifugal filter unit (Centricon^® or Microcon^®).

Centrifugal filter units are used to purify and concentrate DNA. When extracting DNA from small or degraded forensic samples, the final concentration of DNA may be too low for subsequent amplification. Although 1ng is a target quantity of DNA for amplification, if 1ng is suspended in 100 µl of fluid, it would be impossible to transfer 100 µl of this solution into an amplification reaction optimized for 50 µl or less. Centrifugal filter units increase the concentration of DNA in solution by retaining the DNA while eliminating a portion of the fluid from the sample. Another benefit of the unit is the ability to secure DNA while contaminants (possibly PCR inhibitors) are washed from the sample.

Centrifugal filter units separate molecules by size through a series of washing and centrifugation steps. The Millipore Corporation produces two centrifugal filter units under the names Centricon^® and Microcon^®.

Attributes of Centricon^® and Microcon^® filter units include:

Both employ Amicon^® filters to retain the DNA.
Filter porosity varies.
Proteins flow through, rather than sticking to, the surface during centrifugation.
Filters are anisotrophic, with increasingly smaller interstitial spaces in the direction of filtration, allowing for better retention of smaller molecules.
Filters are composed of regenerated cellulose, which can be sterilized.
Filters exhibit sufficient strength when wet.

Note:
It is important not to spin the unit at rates higher than recommended by the manufacturer because the unit may become compromised, resulting in reduced sample recovery.

Textile dye molecules, such as indigo, are known PCR inhibitors and are readily washed through the filter. Salts introduced by buffers are removed from the sample during the process. Salts carried over from the process may interfere with capillary electrophoresis.

Read about capillary electrophoresis in course: Amplified DNA Product Separation.

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**Note:**
Care must be taken to avoid introducing phenol from the organic extraction into the unit because it will break down both the cellulose filter and its supporting silicone rubber o-ring.

Step Four Reagents

The only reagents used in this process are buffers used in the DNA purification process.

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Date Created: July 6, 2023