[Prime-Editing Methods and pegRNA Design Programs].

It has been 10 years since CRISPR/Cas technology was applied to edit the genomes of various organisms. Its ability to produce a double-strand break in a DNA region specified by the researcher started a revolution in bioengineering. Later, the Base Editing (BE) method was developed. BE is performed via the formation of single-strand breaks by the mutant form of Cas nuclease (nickase), fused with deaminases and other enzymes. It can be used to promote A ↔ G and C ↔ T transitions, and a C → G transversion. Just over 3 years ago, a new Prime Editing (PE) variant of CRISPR/Cas was invented. Unlike BE, in PE the nickase is fused with reverse transcriptase, capable of building a new DNA chain using the pegRNA template. The pegRNA consists of an elongated guide RNA with an extra sequence at the 3'-end. Prime editing makes it possible to insert the desired mutations into this extra sequence and to carry out any substitutions and indels of bases without the use of special donor DNA. To date, a number of PE variants have been proposed; they are briefly considered in this review with an emphasis on prime editing of plant genomes. Some attention is also paid to pegRNA design programs, as well as evaluation of the efficiency of the editing. Such a variety of PE techniques is due to the opportunities of high-precision introduction of desired changes with a rather low frequency of off-target mutations in the genomes of various organisms. The relatively low efficiency of prime editing inspires researchers to offer new approaches. There is hope that further development of the technology will improve PE enough to take its rightful place among the genome targeting methods that are suitable for any organisms, and will have a positive impact on the agricultural sector, industrial biotechnologies, and medicine.

[New polymorphic DNA marker to determine a person's sex from biological material]. / Novyi polimorfnyi DNK-marker dlya opredeleniya pola cheloveka po ego biologicheskomu materialu.

The objective of the study was to pre-evaluate the applicability of gender-specific nucleotide sequences in human neuroligin genes as alternative DNA markers of sex. A new polymorphic locus based on NLGNX and NLGNY genes was proposed to establish the sex attribute of human biomaterials. The significant difference in the location of these loci relative to the pseudoautosomal region (PAR), as well as the combination of different types of polymorphism on the one hand, and the possibility of using gender-specific primers «in one assay¼ on the other hand, warrants their use as an additional marker of human sex attribute, including utilization as part of systems for DNA registration in the population. The introduction of a new polymorphic locus based on the NLGNX and NLGNY genes will make it possible to reliably identify the sex attribute of biological material recovered from crime scenes.

Rolling Circle Amplification as a Universal Method for the Analysis of a Wide Range of Biological Targets.

Detection and quantification of biotargets are important analytical tasks, which are solved using a wide range of various methods. In recent years, methods based on the isothermal amplification of nucleic acids (NAs) have been extensively developed. Among them, a special place is occupied by rolling circle amplification (RCA), which is used not only for the detection of a specific NA but also for the analysis of other biomolecules, and is also a versatile platform for the development of highly sensitive methods and convenient diagnostic devices. The present review reveals a number of methodical aspects of RCA-mediated analysis; in particular, the data on its key molecular participants are presented, the methods for increasing the efficiency and productivity of RCA are described, and different variants of reporter systems are briefly characterized. Differences in the techniques of RCA-mediated analysis of biotargets of various types are shown. Some examples of using different RCA variants for the solution of specific diagnostic problems are given.

[Synthesis of Circular DNA Templates with T4 RNA Ligase for Rolling Circle Amplification].

Currently, isothermal methods of nucleic acid amplification have been well established; in particular, rolling circle amplification is of great interest. In this approach, circular ssDNA molecules have been used as a target that can be obtained by the intramolecular template-dependent ligation of an oligonucleotide C-probe. Here, a new method of synthesizing small circular DNA molecules via the cyclization of ssDNA based on T4 RNA ligase has been proposed. Circular ssDNA is further used as the template for the rolling circle amplification. The maximum yield of the cyclization products was observed in the presence of 5-10% polyethylene glycol 4000, and the optimum DNA length for the cyclization constituted 50 nucleotides. This highly sensitive method was shown to detect less than 10^(2) circular DNA molecules. The method reliability was proved based on artificially destroyed dsDNA, which suggests its implementation for analyzing any significantly fragmented dsDNA.

[PCR-based evaluation of sequence specificity of DNA fragmentation by ultrasound].

Ultrasonic fragmentation, which is a simple and convenient method for the mechanical degradation of DNA, is widely used in modern genome studies as one of the sample preparation steps. It has been recently found that the DNA breaks occur more often in the regions containing 5'-CG-3' dinucleotides. We studied the influence of the 5'-CG-3' dinucleotides on the efficiency of the 28S rRNA gene amplification during PCR with sonicated DNA of Mantis religiosa. It was shown that the amplification rate depends on the template length and the number of 5'-CG-3' dinucleotides. Amplification of the DNA regions with a higher 5'-CG-3' density is less efficient because of their higher sensitivity to ultrasound. The amount of the amplified DNA templates is inversely proportional to the 5'-CG-3'number.

[The Increase of Oligonucleotides--Gold Nanoparticles Conjugates Stability].

For today the reagents based on oligonucleotides--gold nanoparticles conjugates and used for specific nucleic acids detection are actively being developed. Such molecular structures are stabilized through the bonds between thiol group in oligonucleotides and gold atoms in nanoparticle. The durability of oligonucleotides--gold nanoparticles binding affects directly on the stability of conjugates and on the possibility of further manipulations. In this paper, a method for the strengthening of oligonucleotides attachment on the gold nano-particles surface by means of anchor groups with dithiolane residues is proposed. A comparative study of the anchors molecular structure influence on the conjugates stability at conditions that typical for oligonucleotide probes was carried out.

[DNA amplification using PCR with abutting primers].

DNA analysis of ñîmplex biological objects (wastewater, soil, archaeological and forensic samples, etc.) is currently of great interest. DNA of these objects is characterized by low suitability for research due to the violation of its integrity and chemical structure; thus, the detection of specific nucleic acid fragments can be achieved by PCR with contiguous primers. In this paper, we present the results that clarify the specific characteristics of PCR with abutting primers. The 3'-ends of these primers are annealed at adjacent nucleotides of complementary chains of DNA target. It has been shown that the proximity of primers enables the formation of specific reaction products with a higher sensitivity and less reaction time. Using artificially damaged DNA and DNA from the soil we demonstrated that the abutting primers provide assured detection of specific DNA fragments. The results of this work may be taken into account in PCR with degraded (fragmented) DNA.

[New approaches to the real-time detection of nucleotide mismatches by means of chimeric hybridization probes].

New approaches to the detection of impaired nucleotides based on the allele specific ligation of a "C probe" followed by rolling circle amplification have been developed. The detection of amplification products was realized by using enzymatic and deoxyribozyme digestion of fluorescently-labeled DNA-RNA-DNA chimeric oligonucleotide structures in cycling probe technology (CPT) in real-time mode.