[Preparation of prokaryotic cDNA for high-throughput transcriptome analysis].

High contents of non-coding RNA in total bacteria RNA complicates considerably transcriptome analysis using standard approaches like high-throughput sequencing, gene expression profiles, subtractive hybridization. We suggest a procedure of preparation of bacterial cDNA for transcriptomics that includes rRNA and tRNA depletion with preservation of relative abundance of coding sequences. The method is based on the second order hybridization kinetics and unique properties of Kanchatka crab duplex-specific nuclease.

Normalization of full-length-enriched cDNA.

A well-recognized obstacle to efficient high-throughput analysis of cDNA libraries is the differential abundance of various transcripts in any particular cell type. Decreasing the prevalence of clones representing abundant transcripts before sequencing, using cDNA normalization, may significantly increase the efficacy of random sequencing and is essential for rare gene discovery. Duplex-specific nuclease (DSN) normalization allows the generation of normalized full-length-enriched cDNA libraries to permit a high gene discovery rate.

[Identification of the amino acid residues responsible for the reversible photoconversion of the monomeric red fluorescent protein TagRFP protein].

The site-directed mutagenesis of the monomeric red fluorescent protein TagRFP and its variants was performed with the goal of generating reversibly photoactivatable fluorescent proteins. Amino acids at positions 69, 148, 165, 179, and 181 (enumeration according to the green fluorescent protein GFP) were shown to play a key role in the manifestation of the photoactivatable properties. A reversibly photoactivatable red fluorescent protein KFP-HC with excitation and emission maxima at 585 and 615 nm, respectively, was generated.

DSN depletion is a simple method to remove selected transcripts from cDNA populations.

A novel DSN-depletion method allows elimination of selected sequences from full-length-enriched cDNA libraries. Depleted cDNA can be applied for subsequent EST sequencing, expression cloning, and functional screening approaches. The method employs specific features of the kamchatka crab duplex-specific nuclease (DSN).

Isolation, characterization and molecular cloning of duplex-specific nuclease from the hepatopancreas of the Kamchatka crab.

Background: Nucleases, which are key components of biologically diverse processes such as DNA replication, repair and recombination, antiviral defense, apoptosis and digestion, have revolutionized the field of molecular biology. Indeed many standard molecular strategies, including molecular cloning, studies of DNA-protein interactions, and analysis of nucleic acid structures, would be virtually impossible without these versatile enzymes. The discovery of nucleases with unique properties has often served as the basis for the development of modern molecular biology methods.