No abstract

DNA polymerases synthesize new DNA strands according to the template DNA, using deoxynucleotide triphosphates during DNA replication and repair, and are essential to maintain genome integrity in DNA metabolism. In addition, these enzymes are widely used for genetic engineering techniques, including dideoxysequencing, PCR, DNA labeling, mutagenesis, and other in vitro experiments. Thermostable DNA polymerases are especially useful for PCR and cyclesequencing. We propose a powerful strategy using environmental DNA as a genetic resource to investigate the structure-function relationships of the family B DNA polymerases. The region corresponding to the active center of the DNA polymerizing reaction in the structural gene of P. furiosus DNA polymerase I (PolBI) was substituted by PCR fragments amplified from DNAs within soil samples from various locations in Japan. The chimeric pol genes were constructed within the PolBI expression plasmid. The chimeric enzymes thus produced revealed DNA polymerase activities with different properties.

A quantitative description of glyco-alteration/ differences in diseases can lead to the development of a diagnostic agent for use in vitro to monitor the degree of change in target glycoproteins. Analytical systems have been developed along with the progress of omics-oriented technologies. For clinical implementation, their full automation is required with an apparatus that is simple to operate. Here, we report an automatic analysis system for quantitative characterization of glycoalteration/differences that depends on the unique strategy of "bead arrays in a single tip." The alternative lectin array can obtain a minimum characterization of the glycan profile for nanogram quantities of an endogenous glycoprotein. A simple autopipetting robot produces the precise chemiluminescence detection of glycan−lectin interactions with a wide dynamic range that is superior to fluorescence-based lectin arrays. The tip-based array format enables automatic glycan profiling from sample pretreatment to detection with low variation and linear detection, which may facilitate the use of this lectin array in clinical practice.