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Purification of PCR-Inhibitory Components by Cellulose Acetate Membranes

H. Mohamed, D.H. Szarowski, L.A. Lepak, M.G. Spencer, D.L. Martin, M. Caggana and J.N. Turner
New York State Department of Health, US

Keywords:
cellulose acetate, lab-on-a-chip, filtration, DNA purification

Abstract:
Conventional methods for extracting DNA from blood results in the presence of heme, which is an inhibitory factor decreasing PCR sensitivity. Current techniques for removing the inhibitors are laborious, costly, and/or sample-specific. We investigated using electrophoresis and a biopolymer membrane to separate DNA from heme. These membranes are biocompatible, can be cast directly onto silicon wafers using a standard microfabrication process without use of adhesives or introduction of exogenous contaminants. We have characterized the membranes’ properties and altered their casting conditions yielding a filter with a low molecular weight cut-off: 350Da. Purified genomic DNA was mixed with hemoglobin, and the mixture was boiled to denature the DNA and dissociate the hemoglobin releasing heme into solution. The solution was immediately cooled, and placed in a chamber on one side of the membrane. By applying an electric field across this membrane, and running the experiment on ice, we were able to pass the single-stranded DNA while restricting heme since its formula weight (614Da) is larger than the membrane cutoff. The DNA was purified and enriched as observed by spectrophotometry. These membranes are easy to make, inexpensive, and can be incorporated into existing DNA chips, thereby adding a purification module to create a lab-on-a-chip design.

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