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DNA-free A NEW Method to Remove DNA

can be designed to control for genomic DNA contamination. Primers that span intron-exon boundaries amplify a product from contaminating DNA that includes the intron, making it much larger than the expected cDNA product. In fact, primers can be designed to span a genomic fragment large enough to make amplification from genomic DNA effectively impossible. Relying solely on primer design for the detection of DNA contamination, however, is not always enough. Pseudogenes may exist in your sample that can produce an amplified product of the same size as the expected cDNA product. (Pseudogenes arise from a processed mRNA that is reverse transcribed and then integrated into the genome; no introns are present). Figure 2 illustrates how primer design can be used to detect most DNA contamination, and why a "minus-RT" control remains necessary in any RT-PCR experiment.

Figure 2. Detection of DNA Contamination in RNA. Mouse liver RNA (0.5 g) was used in RT-PCR. The S15 PCE primers span an intron-exon junction. Products from both the gene and the pseudogene for this message are detected in the minus-RT reaction.


Getting Rid of Contaminating DNA and the DNase Used to Destroy it

Because virtually all RNA samples have trace amounts of contaminating DNA, most protocols specify DNase treatment for RT-PCR applications. DNase I treatment is clearly the best way to rid an RNA sample of contaminating DNA. However, some preparations of DNase may be contaminated with RNases, and the DNase must be completely inactivated prior to RT-PCR so that it doesn't degrade newly synthesized DNA. Unfortunately, removal or
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