Using this artificial base pair system (5-Me-iso-dC:iso-dG, dA:dT*, dC:dG) with KF-Taq, the fidelity in PCR was increased to about 98% per amplification cycle. dT*pairs with dA, but not with iso-dG (5). More recently, Sismour and Benner solved this problem by using 2-thio-dT (dT*) in place of dT. The limit in fidelity is chiefly due to the ability of iso-dG’s 1,2 tautomer to mis-pair with dT. In 2004, Johnson and co-workers observed that, by using the Klenow fragment of Taq polymerase (KF-Taq) in PCR, the fidelity of the 5-Me-iso-dC:iso-dG base pair was about 96% per amplification cycle (4). Such optimization is necessary to enable the full development of artificially expanded genetic systems utilizing an expanded genetic code, thereby allowing for the site-specific incorporation of novel functional components (such as unnatural amino acids) into proteins. (b) qPCR and artificially expanded genetic systems: A number of research groups have been working on optimizing PCR amplification on templates containing 5-Me-iso-dC. The limits of detection of the assay were improved 10-fold, from < 500 HIV molecules/mL to < 50 molecules/mL. Use of this strategy resulted in a significant reduction in non-specific hybridization of the above three sequence types to non-target nucleic acid sequences, and thus less amplification of background. For example, Collins and co-workers significantly improved the sensitivity of a branched DNA quantitative hybridization assay for detecting the HIV POL sequence by incorporating ~30% 5-Me-iso-dC and iso-dG into the pre-amplifier, branched DNA (bDNA) amplifier and alkaline phosphate probe sequences used in the assay (3). (a) Molecular recognition: The 5-Me-iso-dC:iso-dG base pair has been incorporated into hybridization assays to enhance probe-target specificity and reduce spurious hybridization to non-target sequences. The combination of 5-Me-iso-dC’s high selectivity for iso-dG, and the resulting base pair’s high thermodynamic stability, make this modified base particular attractive in the following applications: Furthermore, since iso dC and 5-Me-iso-dC does not pair with dG, iso dC and 5-Me-iso-dC:iso-dG can function as a stable unnatural base pair that can be used to expand the genetic code. Substitution of a 5-me iso-dC:iso-dG base pair for a C:G pair increases the Tm of the resulting duplex by ~2degC per base pair substitution (1,2). USA 80, 1830–4.Iso dC and 5-methyl iso-deoxycytosine (5-Me-iso-dC) forms a Watson-Crick base pair with iso-dG, but has a different type of hydrogen bonding pattern than those observed for the natural base pairs A:T and C:G. et al., eds., John Wiley and Sons, New York, NY. (1987) In: Current Protocols in Molecular Biology, Ausubel, F.M. Ĭhoose Your Configuration: Learn more about our custom options for this product at: References The DNA-dependent DNA polymerase is provided with 10X Reaction Buffer. The 3´→5´ exonuclease activity can be used to generate blunt ends from a 3´-overhang. The 5´→3´ polymerase activity of Klenow Fragment can be used to fill in 5´-protruding ends with unlabeled or labeled dNTPs, to sequence single- or double-stranded DNA templates, for in vitro mutagenesis using synthetic oligonucleotides, for cDNA second-strand synthesis and to generate single-stranded DNA probes. coli DNA Polymerase I that lacks the 5´→3´ exonuclease activity of intact DNA polymerase I but retains its 5´→3´ polymerase, 3´→5´ exonuclease and strand displacement activities. DNA Polymerase I Large (Klenow) Fragment is a 68kDa C-terminal fragment of E.
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