Research Update
CleanAmp™ Primers in Action
We first met John Owen, MD of Wake Forest University in early 2009 and learned that he was having some issues with qPCR specificity. His work involved developing a highly sensitive test for a mutation known to cause polycythemia and thought to play a strong role in essential thrombocytosis. This test had to be highly sensitive and specific since the mutation is only present in a small fraction of cells. John received a TriLink ResearchReward to replace the standard primers in his assay with CleanAmp™ Primers. And indeed, John found that CleanAmp™ Primers routinely identified the presence of mutation fractions as low as 0.001%. John stated, “It has been a pleasure dealing with everyone at TriLink. The ResearchRewards program is a great idea and certainly has put TriLink at the top of my list.”
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Technology Spotlight
ResearchRewards for Custom Oligonucleotides
Linda Chelico, Assistant Professor Microbiology and Immunology at the University of Saskatchewan, specializes in the APOBEC family of single-stranded DNA deaminases, in particular APOBEC3G and how it functions as part of the dynamic process of HIV reverse transcription. Linda used her ResearchReward to obtain the dye-labeled and base-modified DNA substrates required to test enzyme characteristics such as specific activity, ssDNA binding affinity, enzyme processivity, enzyme polarity, and the ability of the enzyme to alter the ssDNA structure.
Chris Wilds of Concordia University also received a ResearchReward for modified oligonucleotides. He acknowledged, “It’s wonderful that TriLink has such a program in place. As a young investigator, funds are tight and a program such as this is a great source of support. Also, TriLink kept us apprised throughout the synthesis and offered support and follow up whenever we had questions.”
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Ask An Expert Featured Question
Q - Are there dNTPs that I can use in a PCR to induce random mutations? -Daniel Turner
A - There are a number of dNTP analogs which have been reported to introduce mutations in PCR. One of the most studied set of analogs includes dPTP (cat # N-2037) and 8-oxo-dGTP (cat # N-2034). When used in equimolar concentration to dATP, dCTP, dGTP and dTTP, a number of transition and transversion mutations can be introduced. For further experimental details on the use of these analogs in PCR, please refer to the following references from Zaccolo et al.:
- J. Mol. Biol. (1999) 285, 775-783.
- J. Mol. Biol. (1996) 255, 589-603.
Other mutagenic dNTPs which have been used in PCR mutagenesis schemes include:
- dITP (cat # N-2012): Nucleic Acids Res. (1993) 21, 777-778.
- 5-bromo-dUTP (cat # N-2008): Mol. Biol. Rep. (2008) 35, 663-667.
- 2-hydroxy-dATP: Biol. Pharm. Bull. (2004) 27, 621-623.
PCR random mutagenesis schemes are performed by using modified dNTPs in a first PCR to introduce the mutations, followed by a second PCR using the natural dNTPs. For each of the analogs which have been described, the ratio of modified dNTPs relative to the natural dNTPs was optimized. Therefore, please refer to the primary references listed above for the experimental conditions that are specific for a given dNTP analog.
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