"Why Cy dUTPs and not Cy dTTPs?"

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Tom in Roux Jun-19-00, 09:07 AM (PDT) "Why Cy dUTPs and not Cy dTTPs?" Reviewing the protocols shows that labeling the cDNAs uses cyanine dUTP. It would seem to me that since it's DNA one would use dTTPs. Is it that the flourophore attachment site is where the methyl group is in thymine? Or that the reverse transcriptase has low discrimination between UTP and TTP? As long as it works, I suppose it doesn't matter, but I always like to understand what I'm doing! Thanks for any help. Tom (in the Roux lab at UTAustin)   Alert Edit | Reply | Reply With Quote | Top

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RE: Why Cy dUTPs and not Cy dTTPs?, sps101, Jun-26-00, (1) RE: Why Cy dUTPs and not Cy dTTPs?, dchingcu, Jul-14-00, (2)

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sps101 Jun-26-00, 02:02 AM (PDT) 1. "RE: Why Cy dUTPs and not Cy dTTPs?" When we started, we had the same question, however, we noticed that people were either using dCTP, or dUTP. When we asked around, there did not appear to be any real reason for the choice - just personal preference. I use dCTP because, as you say, you're making cDNA, so it seems more logical to incorporate dCTP than dUTP, but I guess using dTTP would just be another option along the same lines. Good luck. Steve Spencer   Remove | Alert Edit | Reply | Reply With Quote | Top

dchingcu Jul-14-00, 06:37 PM (PDT) 2. "RE: Why Cy dUTPs and not Cy dTTPs?" I am new to the microarray business myself and have asked the same question. Based on the info from Molecular Probes Handbook re the structure of the ChromaTide nucleotides (see below), I have decided to try and use Cy dUTPs. I hope this helps. "Our ChromaTide nucleotides are modified at the C-5 position of uridine via a unique aminoalkynyl linker (Figure 8.4). The C-5 position of UTP or dUTP is not involved in Watson–Crick base-pairing and so interferes little with probe hybridization. In contrast, labeled dATP and dCTP nucleotides are generally modified at the C-6 of adenine or the C-4 of cytosine, which are positions directly involved in base pairing. The aminoalkynyl linker between the fluorophore and the nucleotide in our ChromaTide nucleotides is designed to reduce the fluorophore's interaction with enzymes or target binding sites......." Debbie Laudencia-Chingcuanco USDA-WRRC   Remove | Alert Edit | Reply | Reply With Quote | Top

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