We have characterized a novel nuclease from the Kamchatka crab, designated duplex-specific nuclease (DSN). DSN displays a strong preference for cleaving double-stranded DNA and DNA in DNA-RNA hybrid duplexes, compared to single-stranded DNA. Moreover, the cleavage rate of short, perfectly matched DNA duplexes by this enzyme is essentially higher than that for nonperfectly matched duplexes of the same length. Thus, DSN differentiates between one-nucleotide variations in DNA. We developed a novel assay for single nucleotide polymorphism (SNP) detection based on this unique property, termed “duplex-specific nuclease preference” (DSNP). In this innovative assay, the DNA region containing the SNP site is amplified and the PCR product mixed with signal probes (FRET-labeled short sequence-specific oligonucleotides) and DSN. During incubation, only perfectly matched duplexes between the DNA template and signal probe are cleaved by DSN to generate sequence-specific fluorescence. The use of FRET-labeled signal probes coupled with the specificity of DSN presents a simple and efficient method for detecting SNPs. We have employed the DSNP assay for the typing of SNPs in methyltetrahydrofolate reductase, prothrombin and p53 genes on homozygous and heterozygous genomic DNA.[Supplemental material is available online atwww.genome.org. The sequence data from this study have been submitted to GenBank/EMBL/Date Bank under accession nos. AF520591. The following individuals kindly provided reagents, samples, or unpublished information as indicated in the paper: N.K. Yankovsky, A.V. Polyakov, and G.N. Rudenskaya.]

The diversity, architecture, and dynamics of the TCR repertoire largely determine our ability to effectively withstand infections and malignancies with minimal mistargeting of immune responses. In this study, we have employed deep TCRβ repertoire sequencing with normalization based on unique molecular identifiers to explore the long-term dynamics of T cell immunity. We demonstrate remarkable stability of repertoire, where approximately half of all T cells in peripheral blood are represented by clones that persist and generally preserve their frequencies for 3 y. We further characterize the extremes of lifelong TCR repertoire evolution, analyzing samples ranging from umbilical cord blood to centenarian peripheral blood. We show that the fetal TCR repertoire, albeit structurally maintained within regulated borders due to the lower numbers of randomly added nucleotides, is not limited with respect to observed functional diversity. We reveal decreased efficiency of nonsense-mediated mRNA decay in umbilical cord blood, which may reflect specific regulatory mechanisms in development. Furthermore, we demonstrate that human TCR repertoires are functionally more similar at birth but diverge during life, and we track the lifelong behavior of CMV- and EBV-specific T cell clonotypes. Finally, we reveal gender differences in dynamics of TCR diversity constriction, which come to naught in the oldest age. Based on our data, we propose a more general explanation for the previous observations on the relationships between longevity and immunity.

Suppression subtractive hybridization (SSH) is one of the most powerful and popular methods for isolating differentially expressed transcripts. However, SSH-generated libraries typically contain some background clones representing non-differentially expressed transcripts. To overcome this problem we developed a simple procedure that substantially decreases the number of background clones. This method is based on the following difference between target and background cDNAs: each kind of background molecule has only one orientation with respect to the two different flanking adapter sequences used in SSH, while truly differentially expressed target cDNA fragments are represented by both sequence orientations. The described method selects the molecules that arose due to hybridization of such mirror-orientated molecules. The efficiency of this method was demonstrated in both model and real experimental subtractions.

Introduction. The human gut microbiota is currently seen as an important factor that can promote autism spectrum disorder (ASD) development in children. Aim. This study aimed to detect differences in the taxonomic composition and content of bacterial genes encoding key enzymes involved in the metabolism of neuroactive biomarker compounds in the metagenomes of gut microbiota of children with ASD and neurotypical children. Methodology. A whole metagenome sequencing approach was used to obtain metagenomic data on faecal specimens of 36 children with ASD and 21 healthy neurotypical children of 3–5 years old. Taxonomic analysis was conducted using MetaPhlAn2. The developed bioinformatics algorithm and created catalogue of the orthologues were applied to identify bacterial genes of neuroactive compounds in the metagenomes. For the identification of metagenomic signatures of children with ASD, Wilcoxon's test and adjustment for multiple comparisons were used. Results. Statistically significant differences with decreases in average abundance in the microbiota of ASD children were found for the genera Barnesiella and Parabacteroides and species Alistipes putredinis , B. caccae , Bacteroides intestinihominis, Eubacterium rectale , Parabacteroides distasonis and Ruminococcus lactaris . Average relative abundances of the detected genes and neurometabolic signature approach did not reveal many significant differences in the metagenomes of the groups that were compared. We noted decreases in the abundance of genes linked to production of GABA, melatonine and butyric acid in the ASD metagenomes. Conclusion. For the first time, the neurometabolic signature of the gut microbiota of young children with ASD is presented. The data can help to provide a comparative assessment of the transcriptional and metabolomic activity of the identified genes.

The registration of the accumulation of polymerase chain reaction (PCR) products in the course of amplification (real-time PCR) requires specific equipment, i.e., detecting amplifiers capable of recording the level of fluorescence in the reaction tube during amplicon formation. When the time of the reaction is complete, researchers are able to obtain DNA accumulation graphs. This review discusses the most promising algorithms of the analysis of real-time PCR curves and possible errors, caused by the software used or by operators’ mistakes. The data included will assist researchers in understanding the features of a method to obtain more reliable results.