carbocyanines and 2--deoxycytidine-5--triphosphate

Thermophilic DNA polymerases of the polB family are of great importance in biotechnological applications including high-fidelity PCR. Of particular interest is the relative promiscuity of engineered versions of the exo- form of polymerases from the Thermo- and Pyrococcales families towards non-canonical substrates, which enables key advances in Next-generation sequencing. Despite this there is a paucity of structural information to guide further engineering of this group of polymerases. Here we report two structures, of the apo form and of a binary complex of a previously described variant (E10) of Pyrococcus furiosus (Pfu) polymerase with an ability to fully replace dCTP with Cyanine dye-labeled dCTP (Cy3-dCTP or Cy5-dCTP) in PCR and synthesise highly fluorescent "CyDNA" densely decorated with cyanine dye heterocycles. The apo form of Pfu-E10 closely matches reported apo form structures of wild-type Pfu. In contrast, the binary complex (in the replicative state with a duplex DNA oligonucleotide) reveals a closing movement of the thumb domain, increasing the contact surface with the nascent DNA duplex strand. Modelling based on the binary complex suggests how bulky fluorophores may be accommodated during processive synthesis and has aided the identification of residues important for the synthesis of unnatural nucleic acid polymers.

Topics: Amino Acid Sequence; Amino Acid Substitution; Apoenzymes; Archaeal Proteins; Carbocyanines; Catalytic Domain; Conserved Sequence; Crystallography, X-Ray; Deoxycytosine Nucleotides; DNA; DNA Polymerase beta; Evolution, Molecular; Fluorescent Dyes; Models, Molecular; Protein Binding; Protein Structure, Secondary; Pyrococcus furiosus; Staining and Labeling; Structural Homology, Protein; Substrate Specificity

Here, we describe a method for directly transferring very small amounts of reaction products from one surface to another. The approach is illustrated using a T4 DNA polymerase reaction to extend primers hybridized to a surface-confined DNA template. Following the extension reaction, the resulting oligonucleotide is transferred to a product surface. The important results are that (1) the spatial registration of the product is preserved after transfer; (2) the same reactant surface can be used to generate and transfer multiple iterations of products; and (3) the reaction products are biologically active after transfer.

Topics: Biotin; Carbocyanines; Deoxycytosine Nucleotides; Dimethylpolysiloxanes; DNA-Directed DNA Polymerase; DNA, Single-Stranded; Fluorescent Dyes; Microscopy, Fluorescence; Streptavidin; Surface Properties; Viral Proteins

Fluorescence resonance energy transfer (FRET) is one of the most powerful and promising tools for single nucleotide polymorphism (SNP) genotyping. However, the present methods using FRET require expensive reagents such as fluorescently labeled oligonucleotides. Here, we describe a novel and cost-effective method for SNP genotyping using FRET. The technique is based on allele-specific primer extension using mononucleotides labeled with a green dye and a red dye. When the target DNA contains the sequence complementary to the primer, extension of the primer incorporates the green and red dye-labeled nucleotides into the strand, and red fluorescence is emitted by FRET. In contrast, when the 3' end nucleotide of the primer is not complementary to the target DNA, there is no extension of the primer, or FRET signal. Therefore, discrimination among genotypes is achieved by measuring the intensity of red fluorescence after the extension reaction. We have validated this method with 11 SNPs, which were successfully determined by end-point measurements of fluorescence intensity. The new strategy is simple and cost-effective, because all steps of the preparation consist of simple additions of solutions and incubation, and the dye-labeled mononucleotides are applicable to all SNP analyses. This method will be suitable for large-scale genotyping.

Topics: Alleles; Carbocyanines; Deoxycytosine Nucleotides; Deoxyuracil Nucleotides; DNA; Fluoresceins; Fluorescence Resonance Energy Transfer; Fluorescent Dyes; Gene Frequency; Genotype; Nucleotides; Polymerase Chain Reaction; Polymorphism, Single Nucleotide; Receptor, Serotonin, 5-HT2A; Reproducibility of Results

Topics: Carbocyanines; Deoxycytosine Nucleotides; DNA, Complementary; Oligonucleotide Array Sequence Analysis