Probe Specific Activity
|The specific activity of nucleic acid probes is an important parameter to control, since it determines the sensitivity of nucleic acid detection. Probe specific activity is not only dependent on the specific activity and amount of radiolabeled nucleotide incorporated into the probe, but also on the amount of probe available for hybridization. Therefore, when choosing a method for synthesis of high specific activity probes, one should take into account the ability of the enzymatic reaction to incorporate low concentrations of high specific activity radiolabeled nucleotides (e.g. 800 Ci/mmol, 10 mCi/ml vs. 6000 Ci/mmol, 10 mCi/ml) and what amount of radiolabeled nucleotide can be economically afforded per reaction. These factors should be balanced with the ability to degrade or separate the template used from the probe synthesized so that the template will not decrease the effective amount of probe available for hybridization. Below, four methods for generating labeled nucleic acids are evaluated for their ability to produce probes of high specific activity, taking into account these criteria.|
In Vitro Transcription of RNA Probes
Higher specific activity labeled nucleotides (e.g. 3000 Ci/mmol) may also be used. However, since they are usually also sold at 10 mCi/ml, the same 50 µCi in a 20 µl reaction volume would result in a lower concentration of limiting nucleotide such that the synthesis of full-length transcripts would be greatly reduced. This problem can be overcome using Ambion's CU Minus™ promoter technology. Transcription reactions containing low concentrations of a nucleotide (e.g. 32P-UTP or 32P-CTP) encoded in the first 12 bases of a transcript experience high levels of abortive transcription. Eliminating C and U nucleotides from the first 12 bases that will be incorporated after the promoter sequence greatly reduces abortive transcription. CU Minus vectors effectively incorporate radiolabeled nucleotides at specific activities of 3000 or 6000 Ci/mmol (without addition of cold nucleotides), which translates into higher specific activity probes (up to 7.5 times higher) and thus stronger signals in nuclease protection assays and blot hybridizations. In addition to CU Minus vectors, Ambion also supplies primers to convert existing T7, T3 and SP6 promoters in any vector to CU Minus promoters.
RNA probes produced can be readily separated from the DNA template by DNase treatment of the terminated transcription reaction and/or gel purification. In addition, probe is generated from only one of the template strands. Therefore, there is no template or second probe strand to effectively lower the probe's specific activity by competing with target for hybridization to it.