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Streptococcus pneumoniae (S. pneumoniae), which is a Gram-positive diplococcus, has emerged as a significant human pathogen. It is a primary cause of bacterial pneumonia, otitis media, meningitis, and septicemia, leading to a considerable impact on global morbidity and mortality. The investigation of S. pneumoniae and its virulence factors has resulted in the identification of surface endonuclease A (EndA). EndA functions in DNA uptake during natural transformation and plays a significant role in gene transfer. The ability of S. pneumoniae to degrade neutrophil extracellular traps (NETs) enhances its virulence and invasive potential in pneumococcal infections. NETosis occurs when neutrophils release chromatin into the extracellular space to form NETs, capturing and neutralizing pathogens. Currently, NETosis can be induced by several microbes, particulate matter, and sterile stimuli through distinct cellular mechanisms, and this includes the involvement of EndA in S. pneumoniae. Here, we reviewed the cellular functions of EndA, its role in S. pneumoniae as a virulence factor in relation to NETosis, its relationship to immunogenicity, and its involvement in several diseases. The discovery of this relationship would significantly impact therapeutic technology in reducing disease burden, especially pneumococcal infections.

The purpose of the research is to apply molecular genetic research methods to identify the taxonomic affiliation of gastrointestinal parasitic sheep nematodes of the family Trichostrongylidae using nested PCR followed by the restriction fragment length polymorphism (RFLP) analysis.Materials and methods. Parasitic nematodes, L3 Strongylata larvae obtained from incubated fecal samples of sheep. The genomic DNA was isolated using a commercial kit for DNA extraction from micro-quantities of tissues (Synthol, Moscow) as per the manufacturer’s guidelines. For DNA amplification, a T-100 Bio-Rad thermal cycler and a commercial Eurogen Master Mix reagent kit were used. The PCR regime was performed according to the WAAVP guidelines, 2006. The restriction endonuclease Rsa I of amplified Trichostrongylidae fragments was performed according to guidelines of the enzyme manufacturer (Sibenzyme, Novosibirsk).Results and discussion. To determine the taxonomic affiliation of Strongylata larvae isolated after incubation of feces from sheep, molecular genetic studies were performed using nested PCR followed by the restriction fragment length polymorphism (RFLP) analysis. This method makes it possible to identify, with the least effort, the genotypes of three species of Strongylata Haemonchus contortus, Trichostrongylus colubriformis, and Teladorsagia circumcincta at the larval stage.

A cornerstone of bacterial molecular biology is the ability to genetically manipulate the microbe under study. Many bacteria are difficult to manipulate genetically, a phenotype due in part to robust removal of newly acquired DNA, for example, by restriction-modification (R-M) systems. Here, we report approaches that dramatically improve bacterial transformation efficiency, piloted using a microbe that is challenging to transform due to expression of many R-M systems, Helicobacter pylori. Initially, we identified conditions that dampened expression of several R-M systems and concomitantly enhanced transformation efficiency. We then identified an approach that would broadly protect newly acquired DNA. We computationally predicted under-represented short DNA sequences in the H. pylori genome, with the idea that these sequences reflect targets of sequence-based surveillance such as R-M systems. We then used this information to modify and eliminate such sites in antibiotic resistance cassettes, creating a stealth version. Modifying antibiotic resistance cassettes in this way resulted in significantly higher transformation efficiency compared to non-modified cassettes, a response that was genomic loci independent. Our results suggest that avoiding R-M systems, via modification of under-represented DNA sequences or transformation conditions, is a powerful method to enhance DNA transformation. Our approach to identify under-represented sequences is applicable to any microbe with a sequenced genome.IMPORTANCEManipulating the genomes of bacteria is critical to many fields. Such manipulations are made by genetic engineering, which often requires new pieces of DNA to be added to the genome. Bacteria have robust systems for identifying and degrading new DNA, some of which rely on restriction enzymes. These enzymes cut DNA at specific sequences. We identified a set of DNA sequences that are missing normally from a bacteriums genome, more than would be expected by chance. Eliminating these sequences from a new piece of DNA allowed it to be incorporated into the bacterial genome at a higher frequency than new DNA containing the sequences. Removing such sequences appears to allow the new DNA to fly under the bacterial radar in stealth mode. This transformation improvement approach is straightforward to apply and likely broadly applicable.

Background and purpose: Lung cancer is one of the deadliest cancers in the world, early detection of lung cancer is one of the biggest challenges in treating this disease and can be very effective in saving patients. Considering the role of microRNAs in the development and progression of lung cancer, the effective factors in the expression and function of microRNAs, including the polymorphisms in the genes of microRNAs, can be used as biomarkers for the early diagnosis of lung cancer. This study aims to investigate the relationship between single nucleotide polymorphisms (rs4919510) of miR608 and (rs6505162) of miR-423 with the risk of lung cancer. Materials and methods: In this case-control study, genotypic analysis of rs6505162 miR-423 and rs4919510 miR-608 polymorphisms was performed on two groups including 110 lung cancer patients and 120 healthy individuals by PCR-RFLP method. After receiving the consent form from the patients, 5 ml of venous blood was collected from each of the study subjects, and the genomic DNA of each person was extracted using the salt precipitation method, the quality of the DNA was measured by absorption spectroscopy, and then all the samples were PCR And by using restriction enzyme digestion by Rsa I and PVUII enzymes, the genotype was determined for both polymorphisms. In the last step, the results of enzyme digestion were checked by electrophoresis on 2% agarose gel, to confirm the RFLP results from sequencing. Random samples were used. Statistical analysis of data was done with SPSS software. Results: In the investigation of mrs6505162 polymorphism in miR-423, the frequency distribution of CC, CA, and AA genotypes in lung cancer patients was 42.7, 34.5, and 22.7, respectively, and in the control group, 30.8, 54.2, 15, respectively which did not have a significant difference. The genotype CA compared to CC and also CA compared to CC+AA was related to the reduction of lung cancer (P=0.009, 95% CI: 0.256-0.829, OR=0.460; P=0.003, 95% CI: 0.262-0.760, OR=0.447) which can indicate the protective role of this genotype. In the investigation of rs4919510 polymorphism in miR608, the frequency of allele G was 16.4% in the patient group and 20.4% in the control group, and the frequency of allele C was 83.6% in the patient group and 79.6% in the control group. This allele frequency was not statistically significant with a probability ratio of 0.763 and a confidence interval of 1.227-0.474 (P=0.263). No significant difference was observed between the group of cancer patients and control subjects. Codominant, Dominant, Over dominant, and Recessive, statistically no significant difference was observed. Conclusion: According to our findings in miR-423, the CA genotype in rs6505162 polymorphism can have a protective role in lung cancer, but rs4919510 polymorphism in miR-608 was not significantly associated with lung cancer, further studies with more samples are suggested to confirm these findings.

Context: Parapatric sister species are ideal for tests of ecological interactions. Pacific (Martes caurina) and American pine (M. americana) martens are economically and culturally valuable furbearers that hybridize in the north-central Rocky Mountains. Despite preliminary evidence of biased introgression, the hybrid zone has been geographically stable for 70 years, but interspecific ecological interactions have yet to be examined in detail. Objectives: We test whether ecological interactions may influence the outcome of hybridization in this system. To that end, we estimate the fundamental niche of each species and gauge how suitability landscapes change when the two species are in contact. Methods: We genotyped > 400 martens from the Rocky Mountain hybrid zone to diagnose individuals to species-level and identify putative hybrids. We then built range-wide ecological niche models for each species, excluding individuals in the hybrid zone, to approximate their respective fundamental niches. Those models were projected into the hybrid zone and compared with niche models trained on individuals within the hybrid zone to assess how niche dynamics change when the species are in sympatry. Results: The fundamental niche of each species differed significantly, while the hybrid zone was equally suitable for both. Niches of each species based on models built within the hybrid zone showed that Pacific martens utilized significantly less suitable habitat than expected based on their range-wide fundamental niche, suggesting that species interactions shape local hybridization. We detected few admixed individuals (12%), with no evidence of directional (sex or species) biases. Interstate-90 further acts as a major dispersal barrier. Conclusions: North American martens are currently managed as a single species by some state agencies, yet significant ecological and genetic differences indicate they should be managed separately. The observed ecological displacement of Pacific martens by American pine martens may partially explain the mixed success of historical, mixed-species wildlife translocations and cautions such translocations in the future. Landscape-scale consideration of ecological dynamics, in addition to molecular compatibility, will be essential to the success of future translocations. [ABSTRACT FROM AUTHOR]

Background and purpose: Lung cancer is one of the deadliest cancers in the world, early detection of lung cancer is one of the biggest challenges in treating this disease and can be very effective in saving patients. Considering the role of microRNAs in the development and progression of lung cancer, the effective factors in the expression and function of microRNAs, including the polymorphisms in the genes of microRNAs, can be used as biomarkers for the early diagnosis of lung cancer. This study aims to investigate the relationship between single nucleotide polymorphisms (rs4919510) of miR608 and (rs6505162) of miR-423 with the risk of lung cancer. Materials and methods: In this case-control study, genotypic analysis of rs6505162 miR-423 and rs4919510 miR-608 polymorphisms was performed on two groups including 110 lung cancer patients and 120 healthy individuals by PCR-RFLP method. After receiving the consent form from the patients, 5 ml of venous blood was collected from each of the study subjects, and the genomic DNA of each person was extracted using the salt precipitation method, the quality of the DNA was measured by absorption spectroscopy, and then all the samples were PCR And by using restriction enzyme digestion by Rsa I and PVUII enzymes, the genotype was determined for both polymorphisms. In the last step, the results of enzyme digestion were checked by electrophoresis on 2% agarose gel, to confirm the RFLP results from sequencing. Random samples were used. Statistical analysis of data was done with SPSS software. Results: In the investigation of mrs6505162 polymorphism in miR-423, the frequency distribution of CC, CA, and AA genotypes in lung cancer patients was 42.7, 34.5, and 22.7, respectively, and in the control group, 30.8, 54.2, 15, respectively which did not have a significant difference. The genotype CA compared to CC and also CA compared to CC+AA was related to the reduction of lung cancer (P=0.009, 95% CI: 0.256-0.829, OR=0.460; P=0.003, 95% CI: 0.262-0.760, OR=0.447) which can indicate the protective role of this genotype. In the investigation of rs4919510 polymorphism in miR608, the frequency of allele G was 16.4% in the patient group and 20.4% in the control group, and the frequency of allele C was 83.6% in the patient group and 79.6% in the control group. This allele frequency was not statistically significant with a probability ratio of 0.763 and a confidence interval of 1.227-0.474 (P=0.263). No significant difference was observed between the group of cancer patients and control subjects. Codominant, Dominant, Over dominant, and Recessive, statistically no significant difference was observed. Conclusion: According to our findings in miR-423, the CA genotype in rs6505162 polymorphism can have a protective role in lung cancer, but rs4919510 polymorphism in miR-608 was not significantly associated with lung cancer, further studies with more samples are suggested to confirm these findings. [ABSTRACT FROM AUTHOR]

Abstract Background Genomic architecture is a key evolutionary trait for living organisms. Due to multiple complex adaptive and neutral forces which impose evolutionary pressures on genomes, there is a huge variability of genomic features. However, their variability and the extent to which genomic content determines the distribution of recovered loci in reduced representation sequencing studies is largely unexplored. Results Here, by using 80 genome assemblies, we observed that whereas plants primarily increase their genome size by expanding their intergenic regions, animals expand both intergenic and intronic regions, although the expansion patterns differ between deuterostomes and protostomes. Loci mapping in introns, exons, and intergenic categories obtained by in silico digestion using 2b-enzymes are positively correlated with the percentage of these regions in the corresponding genomes, suggesting that loci distribution mostly mirrors genomic architecture of the selected taxon. However, exonic regions showed a significant enrichment of loci in all groups regardless of the used enzyme. Moreover, when using selective adaptors to obtain a secondarily reduced loci dataset, the percentage and distribution of retained loci also varied. Adaptors with G/C terminals recovered a lower percentage of selected loci, with a further enrichment of exonic regions, while adaptors with A/T terminals retained a higher percentage of loci and slightly selected more intronic regions than expected. Conclusions Our results highlight how genome composition, genome GC content, RAD enzyme choice and use of base-selective adaptors influence reduced genome representation techniques. This is important to acknowledge in population and conservation genomic studies, as it determines the abundance and distribution of loci.

Abstract Background & Methods In this study, a novel restriction enzyme (RE) digestion-based droplet digital polymerase chain reaction (ddPCR) assay was designed for cg005575921 within the AHRR gene body and compared with matching results obtained by bisulfite conversion (BIS) ddPCR and Illumina DNA methylation array. Results The RE ddPCR cg05575921 assay appeared concordant with BIS ddPCR (r 2 = 0.94, P

Background: Genomic architecture is a key evolutionary trait for living organisms. Due to multiple complex adaptive and neutral forces which impose evolutionary pressures on genomes, there is a huge variability of genomic features. However, their variability and the extent to which genomic content determines the distribution of recovered loci in reduced representation sequencing studies is largely unexplored. Results: Here, by using 80 genome assemblies, we observed that whereas plants primarily increase their genome size by expanding their intergenic regions, animals expand both intergenic and intronic regions, although the expansion patterns differ between deuterostomes and protostomes. Loci mapping in introns, exons, and intergenic categories obtained by in silico digestion using 2b-enzymes are positively correlated with the percentage of these regions in the corresponding genomes, suggesting that loci distribution mostly mirrors genomic architecture of the selected taxon. However, exonic regions showed a significant enrichment of loci in all groups regardless of the used enzyme. Moreover, when using selective adaptors to obtain a secondarily reduced loci dataset, the percentage and distribution of retained loci also varied. Adaptors with G/C terminals recovered a lower percentage of selected loci, with a further enrichment of exonic regions, while adaptors with A/T terminals retained a higher percentage of loci and slightly selected more intronic regions than expected. Conclusions: Our results highlight how genome composition, genome GC content, RAD enzyme choice and use of base-selective adaptors influence reduced genome representation techniques. This is important to acknowledge in population and conservation genomic studies, as it determines the abundance and distribution of loci. [ABSTRACT FROM AUTHOR]

Background & Methods: In this study, a novel restriction enzyme (RE) digestion-based droplet digital polymerase chain reaction (ddPCR) assay was designed for cg005575921 within the AHRR gene body and compared with matching results obtained by bisulfite conversion (BIS) ddPCR and Illumina DNA methylation array. Results: The RE ddPCR cg05575921 assay appeared concordant with BIS ddPCR (r2 = 0.94, P < 0.0001) and, when compared with the Illumina array, had significantly better smoking status classification performance for current versus never smoked (AUC 0.96 versus 0.93, P < 0.04) and current versus ex-smoker (AUC 0.88 versus 0.83, P < 0.04) comparisons. Conclusions: The RE ddPCR cg05575921 assay accurately predicts smoking status and could be a useful component of 'precision-medicine' chronic disease risk screening tools. [ABSTRACT FROM AUTHOR]

In the Triticeae tribe, several subgenomes (St, J, V and others) were described, and a subgenome-specific molecular marker development is an actual branch of studies. Non-transcribed spacers (NTS) of 5S rDNA are perspective to create species-specific or genome-specific molecular markers in closely related organisms. Early, the 5S rDNA NTS based CAPS marker (StJV-f/StJV-r primers with the SmiM1 enzyme digestion) was designed for identification of the St, J and V subgenomes. The V subgenome amplicons were differed from the St and J amplicons in length. The St amplicons were distinguishable from the J amplicons after the SmiM1 enzyme digestion (St fragments were digested, J fragments were not digested, which is a disadvantage, because a failure with the digestion of St amplicons can be mistaken for J amplicons). This article presents the results of the StJV marker improvement. Comparative analysis of restriction enzyme maps revealed that the Hpy166 II enzyme cuts the StJV amplicons of all studied subgenomes in such a way that different product patterns are obtained. Experimental testing confirmed this finding. Thus, the improved CAPS marker can serve as a promising tool for verifying samples in Triticeae plant collections, screening herbarium materials, as well as in the breeding process aimed at improving wheat by involving its wild relatives in traditional breeding programs efficiently. [ABSTRACT FROM AUTHOR]

In recent years, high-throughput sequencing methods have become increasingly popular in molecular biology laboratories, mainly due to the relatively low cost of small, benchtop platforms, the simplicity of library preparation, and the low price per unit of information. Sequencing huge and complex genomes, such as cereal genomes, remains challenging and may not always be necessary. Therefore, several techniques have been developed to sequence a reduced representation of the genome. The most flexible and widely used of these is ddRAD-Seq, which uses a pair of restriction enzymes to generate a pool of DNA fragments. The aim of this study was to validate in vitro the efficacy of different combinations of restriction enzymes for ddRAD-Seq library construction in barley and maize. Eleven pairs of restriction enzymes were selected and tested to determine the concentrations of fragments with the expected length range and to select suitable pairs for sampling the genomes of these two cereals using ddRAD-Seq. For the selected pairs, i.e., PstI—MspI and HindIII—FspBI for barley and maize, respectively, libraries were prepared for NGS sequencing on Illumina MiSeq. Sequencing confirmed the suitability of the selected enzymes to perform ddRAD-Seq in different genotypes. The results presented can be used for extensive research on these important cereal species. [ABSTRACT FROM AUTHOR]

Clostridium carboxidivorans P7 (DSM 15243) is a bacterium that converts syngas (a mixture of CO, H2, and CO2) into hexanol. An optimized and scaled-up industrial process could therefore provide a renewable source of fuels and chemicals while consuming industry waste gases. However, the genetic engineering of this bacterium is hindered by its multiple restriction–modification (RM) systems: the genome of C. carboxidivorans encodes at least ten restriction enzymes and eight methyltransferases (MTases). To gain insight into the complex RM systems of C. carboxidivorans, we analyzed genomic methylation patterns using single-molecule real-time (SMRT) sequencing and bisulfite sequencing. We identified six methylated sequence motifs. To match the methylation sites to the predicted MTases of C. carboxidivorans, we expressed them individually in Escherichia coli for functional characterization. Recognition motifs were identified for all three Type I MTases (CAYNNNNNCTGC/GCAGNNNNNRTG, CCANNNNNNNNTCG/CGANNNNNNNNTGG and GCANNNNNNNTNNCG/CGNNANNNNNNNTGC), two Type II MTases (GATAAT and CRAAAAR), and a single Type III MTase (GAAAT). However, no methylated recognition motif was found for one of the three Type II enzymes. One recognition motif that was methylated in C. carboxidivorans but not in E. coli (AGAAGC) was matched to the remaining Type III MTase through a process of elimination. Understanding these enzymes and the corresponding recognition sites will facilitate the development of genetic tools for C. carboxidivorans that can accelerate the industrial exploitation of this strain. [ABSTRACT FROM AUTHOR]

ABSTRACTA cornerstone of bacterial molecular biology is the ability to genetically manipulate the microbe under study. Many bacteria are difficult to manipulate genetically, a phenotype due in part to robust removal of newly acquired DNA, for example, by restriction-modification (R-M) systems. Here, we report approaches that dramatically improve bacterial transformation efficiency, piloted using a microbe that is challenging to transform due to expression of many R-M systems, Helicobacter pylori. Initially, we identified conditions that dampened expression of several R-M systems and concomitantly enhanced transformation efficiency. We then identified an approach that would broadly protect newly acquired DNA. We computationally predicted under-represented short DNA sequences in the H. pylori genome, with the idea that these sequences reflect targets of sequence-based surveillance such as R-M systems. We then used this information to modify and eliminate such sites in antibiotic resistance cassettes, creating a “stealth” version. Modifying antibiotic resistance cassettes in this way resulted in significantly higher transformation efficiency compared to non-modified cassettes, a response that was genomic loci independent. Our results suggest that avoiding R-M systems, via modification of under-represented DNA sequences or transformation conditions, is a powerful method to enhance DNA transformation. Our approach to identify under-represented sequences is applicable to any microbe with a sequenced genome.IMPORTANCEManipulating the genomes of bacteria is critical to many fields. Such manipulations are made by genetic engineering, which often requires new pieces of DNA to be added to the genome. Bacteria have robust systems for identifying and degrading new DNA, some of which rely on restriction enzymes. These enzymes cut DNA at specific sequences. We identified a set of DNA sequences that are missing normally from a bacterium’s genome, more than would be expected by chance. Eliminating these sequences from a new piece of DNA allowed it to be incorporated into the bacterial genome at a higher frequency than new DNA containing the sequences. Removing such sequences appears to allow the new DNA to fly under the bacterial radar in “stealth” mode. This transformation improvement approach is straightforward to apply and likely broadly applicable.

Linear dichroism (LD) is a differential polarized light absorption spectroscopy used for studying filamentous molecules such as DNA and protein filaments. In this study, we review the applications of LD for the analysis of DNA-protein interactions. LD signals can be measured in a solution by aligning the sample using flow-induced shear force or a strong electric field. The signal generated is related to the local orientation of chromophores, such as DNA bases, relative to the filament axis. LD can thus assess the tilt and roll of DNA bases and distinguish intercalating from groove-binding ligands. The intensity of the LD signal depends upon the degree of macroscopic orientation. Therefore, DNA shortening and bending can be detected by a decrease in LD signal intensity. As examples of LD applications, we present a kinetic study of DNA digestion by restriction enzymes and structural analyses of homologous recombination intermediates, i.e., RecA and Rad51 recombinase complexes with single-stranded DNA. LD shows that the DNA bases in these complexes are preferentially oriented perpendicular to the filament axis only in the presence of activators, suggesting the importance of organized base orientation for the reaction. LD measurements detect DNA bending by the CRP transcription activator protein, as well as by the UvrB DNA repair protein. LD can thus provide information about the structures of protein-DNA complexes under various conditions and in real time. [ABSTRACT FROM AUTHOR]

Coin-operated washing machines are convenient household appliances that are widely used in Thailand. The environment in these devices is suitable for fungal colonization. As a result, they may be a potential risk for yeast infection, particularly opportunistic yeasts such as Candida spp. Thus, the aim of this study was to isolate Candida spp. from thirty coin-operated top load washing machines installed at dormitories near Bansomdejchaopraya Rajabhat University, Bangkok, Thailand. Sterile cotton swabs were used to collect samples from four sites of each device including washing powder drawers, fabric softener drawers, tubs, and lint filters. Samples were inoculated on a surface of Sabouraud dextrose agar (SDA) supplemented with 0.05 g/L chloramphenicol. Polymerase chain reaction – restriction fragment length polymorphism (PCR-RFLP) of ITS1-5.8S-ITS2 regions and MspI as a restriction enzyme were used for Candida spp. identification. We identified several species of Candida spp. including Candida parapsilosis complex, Candida glabrata, Candida tropicalis, Candida haemulonii complex, Candida albicans complex, Candida lusitaniae, and Candida guilliermondii by using PCR-RFLP. This investigation showed that coin-operated washing machines were contaminated with important opportunistic yeasts of Candida spp. Therefore, these devices should be maintained and cleaned regularly. [ABSTRACT FROM AUTHOR]

Bligon goat is one of the crossbred goats raised in Indonesia, which has a prolific nature, potentially increasing the economic benefits. The gene that particularly influences prolific traits is Growth Differentiation Factor 9 (GDF9). This study aims to identify the mutation analysis (SNPs and amino acid changes) and restriction enzymes map in the GDF9 of Bligon goats. Six pairs of primers were used to amplify target sequences of GDF9 by polymerase chain reaction method and continued by sequencing. The sequence products were analyzed to get information on the SNPs and restriction enzyme (RE) around the SNPs for genotyping by PCR-RFLP method. A total of 15 SNPs were found in position g.1956A>C, g.2248G>T, g.2470A>T, g.2172DelA (shift T, Heterozygote), g.2807C>T, g.2919C>T, g.2996C>T, g.3615T>C, g.3855A>C, g.3879A>G, g.3924A>G, g.3943G>T, g.3969G>A, g.3981G>A, and g.4314C>T. Eight out of fifteen SNPs are located at the exon. Thus, the amino acid shows one synonymous at Exon 1 (Leucine to Leucine) and seven non synonymous at exon 2 with varied amino acid alteration (Valine to Alanine, Glutamine to Proline, Lysine to Arginine, Lysine to Arginine, Glutamine to Histidine, Arginine to Lysine, and Serine to Asparagine, respectively). Two SNPs at position g.1956A>C in Exon 1 and g.3855A>C in exon 2 show the homozygote CC and heterozygote AC. The most sample at those two position 67% and 83% homozygote type, respectively. Recognitions of site restriction enzymes BsaI and BsmAI were found at g.1956A>C or g.667C>M (Exon I). SNP g.3855A>C or g.2566C>M was recognized by three restriction enzymes (MspI, HapII, and HpaII). Two SNPs were not recognized by the restriction enzyme, and two other SNPs have more than 12 fragmented sequences, and as a consequence, it is very difficult to analyze the genotype. In conclusion, fifteen site mutations were identified; however, only two potential genetic markers with transversion mutations in Exon 1 and Exon 2 were recognized by restriction enzymes (BsaI, HapII, and MspI). These enzymes were recommended as candidate markers for further genotype identification using the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. [ABSTRACT FROM AUTHOR]

Biomolecules can undergo liquid-liquid phase separation (LLPS), forming dense droplets that are increasingly understood to be important for cellular function. Analogous systems are studied as early-life compartmentalization mechanisms, for applications as protocells, or as drug-delivery vehicles. In many of these situations, interactions between the droplet and enzymatic solutes are important to achieve certain functions. To explore this, we carried out experiments in which a model LLPS system, formed from DNA "nanostar" particles, interacted with a DNA-cleaving restriction enzyme, SmaI, whose activity degraded the droplets, causing them to shrink with time. By controlling adhesion of the DNA droplet to a glass surface, we were able to carry out time-resolved imaging of this "active dissolution" process. We found that the scaling properties of droplet shrinking were sensitive to the proximity to the dissolution ("boiling") temperature of the dense liquid: For systems far from the boiling point, enzymes acted only on the droplet surface, while systems poised near the boiling point permitted enzyme penetration. This was corroborated by the observation of enzyme-induced vacuole-formation ("bubbling") events, which can only occur through enzyme internalization, and which occurred only in systems poised near the boiling point. Overall, our results demonstrate a mechanism through which the phase stability of a liquid affects its enzymatic degradation through modulation of enzyme transport properties.

Advancements in technology over the past few decades have resulted in the development of genome sequencing at lower costs. Protocols, costs, and the amount of data produced by different sequencing technologies are highly variable. Ion Torrent and Illumina sequencing instruments are two sequencing technologies which use very similar library preparation procedures. Enzymatic combinations can be changed in genotyping by sequencing (GbS) library protocols without significant adjustments. To compare the outputs from two different GbS procedures, we sequenced samples of two sister species of yellow-nosed albatross collected at multiple geographic locations. The data sets involving different sequencing instruments and enzymatic combinations were analysed using the Stacks pipeline and aligned to the same reference genome. Both procedures identified the same genetic clusters separating Atlantic and Indian yellow-nosed albatross and substructure within Indian yellow-nosed albatross. [ABSTRACT FROM AUTHOR]

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Clostridium carboxidivorans P7 (DSM 15243) is a bacterium that converts syngas (a mixture of CO, H2, and CO2) into hexanol. An optimized and scaled-up industrial process could therefore provide a renewable source of fuels and chemicals while consuming industry waste gases. However, the genetic engineering of this bacterium is hindered by its multiple restriction–modification (RM) systems: the genome of C. carboxidivorans encodes at least ten restriction enzymes and eight methyltransferases (MTases). To gain insight into the complex RM systems of C. carboxidivorans, we analyzed genomic methylation patterns using single-molecule real-time (SMRT) sequencing and bisulfite sequencing. We identified six methylated sequence motifs. To match the methylation sites to the predicted MTases of C. carboxidivorans, we expressed them individually in Escherichia coli for functional characterization. Recognition motifs were identified for all three Type I MTases (CAYNNNNNCTGC/GCAGNNNNNRTG, CCANNNNNNNNTCG/CGANNNNNNNNTGG and GCANNNNNNNTNNCG/CGNNANNNNNNNTGC), two Type II MTases (GATAAT and CRAAAAR), and a single Type III MTase (GAAAT). However, no methylated recognition motif was found for one of the three Type II enzymes. One recognition motif that was methylated in C. carboxidivorans but not in E. coli (AGAAGC) was matched to the remaining Type III MTase through a process of elimination. Understanding these enzymes and the corresponding recognition sites will facilitate the development of genetic tools for C. carboxidivorans that can accelerate the industrial exploitation of this strain.

Bligon goat is one of the crossbred goats raised in Indonesia, which has a prolific nature, potentially increasing the economic benefits. The gene that particularly influences prolific traits is Growth Differentiation Factor 9 (GDF9). This study aims to identify the mutation analysis (SNPs and amino acid changes) and restriction enzymes map in the GDF9 of Bligon goats. Six pairs of primers were used to amplify target sequences of GDF9 by polymerase chain reaction method and continued by sequencing. The sequence products were analyzed to get information on the SNPs and restriction enzyme (RE) around the SNPs for genotyping by PCR-RFLP method. A total of 15 SNPs were found in position g.1956A>C, g.2248G>T, g.2470A>T, g.2172DelA (shift T, Heterozygote), g.2807C>T, g.2919C>T, g.2996C>T, g.3615T>C, g.3855A>C, g.3879A>G, g.3924A>G, g.3943G>T, g.3969G>A, g.3981G>A, and g.4314C>T. Eight out of fifteen SNPs are located at the exon. Thus, the amino acid shows one synonymous at Exon 1 (Leucine to Leucine) and seven non synonymous at exon 2 with varied amino acid alteration (Valine to Alanine, Glutamine to Proline, Lysine to Arginine, Lysine to Arginine, Glutamine to Histidine, Arginine to Lysine, and Serine to Asparagine, respectively). Two SNPs at position g.1956A>C in Exon 1 and g.3855A>C in exon 2 show the homozygote CC and heterozygote AC. The most sample at those two position 67% and 83% homozygote type, respectively. Recognitions of site restriction enzymes BsaI and BsmAI were found at g.1956A>C or g.667C>M (Exon I). SNP g.3855A>C or g.2566C>M was recognized by three restriction enzymes (MspI, HapII, and HpaII). Two SNPs were not recognized by the restriction enzyme, and two other SNPs have more than 12 fragmented sequences, and as a consequence, it is very difficult to analyze the genotype. In conclusion, fifteen site mutations were identified; however, only two potential genetic markers with transversion mutations in Exon 1 and Exon 2 were recognized by restriction enzymes (BsaI, HapII, and MspI). These enzymes were recommended as candidate markers for further genotype identification using the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method.

Epidemiological studies on amoebic infections are complicated by morphological overlap between the pathogenic E. histolytica, the commensal E. dispar and the amphizoic E. moshkovskii, necessitating molecular identification. The present study developed a simple and economical 18S PCR–RFLP method for the simultaneous detection and differentiation of the three species. PCR products were differentiated by Tat1 restriction digestion generating three different RFLP patterns. Validation was conducted by screening 382 faecal samples from human patients from Kolkata, India, hospitalized for diarrhoea. Analysis indicated that the PCR–RFLP could successfully differentiate between the three species and was confirmed by sequence analysis. This method could prove useful for clinical and epidemiological studies of amoebiasis. [ABSTRACT FROM AUTHOR]

Purpose: The parasites of genera such as Babesia and Theileria are called piroplasmids due to the pear-shaped morphology of the multiplying parasite stages in the blood of the vertebrate host. Because of the enormous number of parasite species and the challenges of multiplex PCR, initial screening of samples using piroplasmid-specific PCR may be a more cost-effective and efficient technique to identify parasite species, especially during epidemiological studies. Accordingly, 18S rRNA PCR was standardized and optimized on common piroplasmids of different animals like cattle, buffaloes, sheep, goats, dogs, horses, and leopards. Methods: Bloods samples from 1250 animals were collected from different animals in Junagadh district of Gujarat, India. 18S rRNA PCR was standardized and optimized as a primary method for molecular screening of piroplasms in domestic and wild animals. The method was checked for its analytical sensitivity and specificity. Parasite species-specific PCR and sequencing was used to validate the test. Moreover, in-silico restriction enzyme (RE) analysis was also done to assess its applicability in PCR–RFLP. Results: Piroplasm infections were recorded in 63.3% of animals in Junagadh. The 18S rRNA PCR detected the piroplasmid DNA in as low as 39 picograms (pg) of whole blood genomic DNA isolated from microscopically Theileria positive blood samples and no reactivity was recorded from common but unrelated haemoparasites viz., Trypanosoma evansi, Hepatozoon spp., Anaplasma spp., and Ehrlichia canis was observed. The 18S rRNA PCR assay findings were confirmed by species-specific PCR and sequencing. Analysis of different sequences generated using 18S rRNA PCR revealed that the amplicon size of Babesia spp. is nearly 400 bp (393–408 bp) whereas Theileria spp. were more than 400 bp (418–424 bp). The percentage of sequence divergence among Babesia and Theileria spp. was 7.3–12.2% and 0.7–12.2%, respectively. In-silico restriction enzyme (RE) analysis reveals the presence of at least one site for a commercially available RE in 18S rRNA fragments of every parasite, which can differentiate it from its congeners. Conclusions: The presented universal oligonucleotide-based PCR assay provides a highly sensitive, specific, cost-effective, and rapid diagnostic tool for the initial screening of piroplasmids infecting domestic and wild animals and is potentially helpful for large-scale epidemiological studies. [ABSTRACT FROM AUTHOR]

Objectives: Periodontitis is an infectious disorder that leads to irreversible loss of the surrounding attachment and bone destruction. Genetic polymorphism of cytokines has been suggested to play a role in periodontitis. This case-control study aimed to investigate the relationship between periodontitis and two single nucleotide polymorphisms (SNPs): rs1800629 (-308 G/A) and rs1799964 (-1031 T/C), in the TNF-α gene promoter area. Materials and methods: Peripheral blood was used to prepare genomic DNA from 60 subjects with stage II to stage III periodontitis, as along with 65 control subjects. Polymerase chain reaction and restriction endonuclease digestion were used to genotype TNF-α SNPs. Results: The distribution of both genotypes and alleles of TNF-α (-308 G/A) polymorphism did not vary between periodontitis subjects and the controls (P > 0.05). However, the CT genotype and C allele of the TNF-α (-1031 T/C) polymorphism were observed more frequently in the periodontitis subjects than in the controls, while the TT genotype and the T allele were more predominant in the control subjects than in the periodontitis patients (OR: 3.149; 95% CI: 1.494–6.639; P = 0.002 and OR: 2.933; 95% CI: 1.413–6.090; P = 0.003, sequentially). Conclusion: The TNF-α (-308 G/A) polymorphism potentially has no correlation with periodontitis susceptibility, whereas the TNF-α (-1031) CT genotype and C allele might be related to periodontitis among Saudi subjects.

In recent years, high-throughput sequencing methods have become increasingly popular in molecular biology laboratories, mainly due to the relatively low cost of small, benchtop platforms, the simplicity of library preparation, and the low price per unit of information. Sequencing huge and complex genomes, such as cereal genomes, remains challenging and may not always be necessary. Therefore, several techniques have been developed to sequence a reduced representation of the genome. The most flexible and widely used of these is ddRAD-Seq, which uses a pair of restriction enzymes to generate a pool of DNA fragments. The aim of this study was to validate in vitro the efficacy of different combinations of restriction enzymes for ddRAD-Seq library construction in barley and maize. Eleven pairs of restriction enzymes were selected and tested to determine the concentrations of fragments with the expected length range and to select suitable pairs for sampling the genomes of these two cereals using ddRAD-Seq. For the selected pairs, i.e., PstI—MspI and HindIII—FspBI for barley and maize, respectively, libraries were prepared for NGS sequencing on Illumina MiSeq. Sequencing confirmed the suitability of the selected enzymes to perform ddRAD-Seq in different genotypes. The results presented can be used for extensive research on these important cereal species.

BMP15-FecX is one of the major genes associated with prolificacy in different sheep breeds (Ovis aries), alongside BMPR15/FecB and GDF9/FecG. The majority of the polymorphisms, associated with ovulation rate in different breeds, are located in the exonic sequences of the gene. In the present study, a fragment of 356 bp, adjacent to the 2nd exon of the BMP-15 gene, was amplified by PCR in 50 individuals of Turcana and Tsigai breeds. The amplified products were subjected to digestion to identify the polymorphisms in the HinfI restriction enzyme sites. The two putative enzyme sites presented in the fragment did not reveal mutations in neither breed, releasing the same electrophoretic profile.

This work includes, identification eight species of Aphids (Homoptera: Aphididae) which collected from the leaves of different plants in many localities of Erbil governorate Kurdistan region-Iraq from the period May till July 2022, these are: Chaitophorus salijaponicus, Aphis fabae, Macrosiphum rosae, Capitophorus carduinus, Myzus persicae, Aphis ruborum, Aphis punca, and Aphis gossypii. The mitochondrial cytochrome C Oxidase subunit I (COI) gene used for identification these species. DNA was isolated, and a band of 550 bp of mt COI gene was amplified during the PCR amplification. The amplicons were digested with HinfI and DdeI restriction enzymes. The restricted fragments produced by RFLP technique were proved by agarose gel electrophoresis. The results illustrated that digested amplicons were given bands according to their cut sites. This study presented that studying aphids to detect their species through a RFLP-PCR technique by using these restriction enzymes can distinguish some species with reliable results. HinfI and DdeI REs could not distinguish all species, HinfI only discriminated species Macrosiphum rosae, Capitophorus carduinus, Myzus persicae and Aphis gossypii, but DdeI identified the remain species, Chaitophorus salijaponicus, Macrosiphum rosae, Myzus persicae and Aphis ruborum, within and among other species exactly. The study suggested using other restriction enzymes to provide full recognition profile for all species. [ABSTRACT FROM AUTHOR]

In silico 16S ribosomal RNA gene-based restriction enzyme approach is a computer-simulated polymerase chain reaction-restriction fragment length polymorphism analysis method, namely customizable in silico sequence evaluation for restriction sites. The purpose of this study was to identify lactic acid bacteria using restriction enzyme analysis of 16S gene (16S ribosomal RNA polymerase chain reaction-restriction fragment length polymorphism). An in silico investigation was conducted to detect species-specific molecular markers in different lactic acid bacteria strains. We selected six out of 20 16S ribosomal RNA gene sequences from probiotic-potential lactic acid bacteria strains. Initially, the corresponding 16S ribosomal RNA gene sequences were selected from the database aligned using the Clustal Omega program and their evolutionary relationships were determined. The results indicated that the studied lactic acid bacteria strains belonged to 6 distinct clades except for the genus of Lactobacillus. Furthermore, the in silico polymerase chain reactionrestriction fragment length polymorphism was conducted for further taxonomic classification. Different restriction enzymes were used to recognize sites present on the lactic acid bacteria 16S ribosomal RNA genes. Customizable in silico sequence evaluation for restriction sites output comprised a predicted agarose gel as virtual polymerase chain reaction-restriction fragment length polymorphism patterns, for proper selection of the appropriate enzyme (s) to be used for polymerase chain reaction-restriction fragment length polymorphism marker generation. Our study demonstrated that ApyPI, CchIII and NheI produced distinct restriction patterns as those of Ku324937_C species. The most abundant species-specific markers were seen for Ku324928_C (10 markers), followed by Ku324908_C (8 markers) of order Hemiptera, then Ku324909_C (5 markers), Ku324898_C (4 markers) and finally, Ku324937_C (3 markers). The results demonstrated that customizable in silico sequence evaluation for restriction sites is a powerful tool to detect species-specific markers to provide better understanding of organisms found in raw and/or fermented milk. [ABSTRACT FROM AUTHOR]

Background and Aim: Toxoplasma gondii is a unicellular coccidian parasite distributed globally and is an important zoonotic pathogen. Approximately 30% of the human population worldwide is chronically infected with T. gondii. The pathogenicity of this species depends on the type originating from the clonal population. Techniques for more accurately determining the type of T. gondii have recently been developed using genetic markers. Specifically, T. gondii has been typed using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). This study aimed to identify sets of PCR-RFLP markers that have high power to discriminate genotyping of T. gondii and are easy to use and are easy to use. The objective of this study was to characterize virulent strain isolates of T. gondii by PCR-RFLP using 10 markers with DdeI. Materials and Methods: T. gondii tachyzoites (RH virulent strain) were derived from culture cells at the Indonesian Research Center for Veterinary Sciences. Genotyping was performed on T. gondii DNA extracted from cell cultured tachyzoites using 10 genetic markers of PCR-RFLP, namely, B1#1, B1#2, B1#3, SAG1#1, SAG1#2, P30, BAG1, ROP1, GRA1, and GRA7, with digestion using the restriction enzyme DdeI. Results: The 10 genes were amplified by PCR. Among them, three genetic markers, B1#3, ROP1, and GRA1, were genotyped by the PCR-RFLP using restriction enzyme DdeI. Overall, the findings showed that the specific RFLP profile of digestion of gene regions by DdeI could be used as a specific marker for the virulent biotype causative of toxoplasmosis. In addition, virulent strains of T. gondii can be easily detected by these markers. Conclusion: Three pairs of primers (B1#3, ROP1, and GRA1) with DdeI have proven useful for the diagnosis of acute toxoplasmosis (virulent strain biotype I). This proposed method is relatively simple, rapid, cheap, and can be performed in most laboratories, providing a practical approach for the routine analysis of T. gondii strains.

MC4R is a gene that has potential effects on growth traits such as body weight and feed intake. The usage of single nucleotide polymorphism (SNPs) in melanocortin-4 receptor (MC4R) as selection markers could help achieve effectiveness in the breeding program. This study aimed to analyze the restriction mapping based on SNPs in the MC4R gene for Bali cattle (Bos sondaicus) compared to various breeds of cattle. Partial MC4R gene was amplified using a primer (F: 5'-ACC AAT GTC AGT GAG TCC CC-3' and R: 5'-CTT CAT GTT GGC GCC CTG-3') with a polymerase chain reaction (PCR) method. Genotype and allele frequencies were calculated using Chi-Square test and analyzed with Hardy-Weinberg law. Restriction enzyme was analyzed using Nebcutter V.2 to see the association between SNPs and the recognition site of restriction enzyme. The result showed four SNPs g.554 T>C, g.634 G>T, g.673 C>T, and g.742 G>A were found in the exon region. SNP g.742 G>A was found as a heterozygote genotype and the rest are SNP g.554 T>C, g.634 G>T, and g.673 C>T were found as homozygote genotypes. All SNPs were synonymous which did not change the amino acid translated. Three restriction enzymes were identified as MmeI, TspRI, and BsrI which attach to SNPs g.554 T>C, g.634 G>T, and g.742 G>A respectively. SNPs found notably g. 742 G>A can be used as genetic markers associated with growth traits for further research on Bali cattle. [ABSTRACT FROM AUTHOR]

[Display omitted] • Successful partial nitritation was conducted under 40 g-NaCL/L. • T-RFLP indicators were developed based on the 16S rRNA gene database. • AOB was discriminated from total bacteria via Tai I and Fnu DII restriction enzymes. • T-RFLP signals improved the R2 of ACR prediction using machine learning by 0.9459. • SHAP proved the contribution of T-RFLP for the ACR prediction in addition to ALR. This study introduces the smart development of a cost-effective biomolecular indicator using terminal restriction fragment length polymorphism (T-RFLP) based on the 16S rRNA gene database obtained from operating the partial nitritation (PN) process under saline conditions. As a result of next-generation sequencing (NGS) on the 16S rRNA gene, ammonia-oxidizing bacteria (AOB) of Nitrosomonas sp. OTU0 (N. OTU0) favored higher salinity of 20–35 g-NaCl/L, while Nitrosomonas sp. OTU8 (N. OTU8) and Nitrosomonas sp. OTU11 (N. OTU11) were predominant under 20 g-NaCl/L. The T-RFLP system with the restriction enzymes of Tai I and Fnu DII, which was thoroughly simulated based on the NGS data of the 16S rRNA gene, effectively differentiates N. OTU0 against N. OTU8 and N. OTU11. In addition, together with AOB's signals, the signals of nitrite-oxidizing and heterotrophic bacteria were obtained as biomolecular indicators of successful PN under saline conditions. These findings suggest that T-RFLP can serve as an economically viable monitoring approach for the sensitive PN process under saline conditions to achieve more sustainable nitrogen removal. For further application, the designated T-RFLP signals of this study were applied to the regression of ammonia conversion rate through machine learning modeling. The regression showed R2 values of 0.9446 and 0.9458 for the training and test sets, respectively. The contribution of T-RFLP for the regression was quantified by a shapley additive explanation (SHAP). [ABSTRACT FROM AUTHOR]

Inclusion Body Hepatitis (IBH) is responsible for huge economic losses to the poultry industry and is caused by Fowl adenoviruses (FAdV). A total of thirty pooled poultry liver homogenates from thirty different poultry farms suspected of IBH have been processed for the PCR-based detection and chicken embryo liver cell culture-based virus isolation. Out of 30 pooled samples, 18 were found positive in PCR; however, only 9 chicken embryo liver cell culture samples having CPE found positive in PCR. The nucleic acid obtained from FAdV specific Polymerase Chain Reaction (PCR) were further genotyped by in-silico restriction enzyme analysis (REA) using enzymes BsiWI, KpnI, and HpaI. Upon phylogenetic analysis, all the nine positive samples of FAdVs clustered in distinct clads of FAdV serotypes 8a, 8b, and 11, which is in correlation with the REA pattern. Thus, nucleic acid sequencing followed by phylogenetic analysis revealed the presence of FAdV-D (serotype 11) and FAdV-E (serotype 8a and 8b) in broiler chickens. [ABSTRACT FROM AUTHOR]

Gene therapy and vaccine development need more novel adenovirus vectors. Here, we attempt to provide strategies to construct adenovirus vectors based on restriction-assembly for researchers with little experience in this field. Restriction-assembly is a combined method of restriction digestion and Gibson assembly, by which the major part of the obtained plasmid comes from digested DNA fragments instead of PCR products. We demonstrated the capability of restriction-assembly in manipulating the genome of simian adenovirus 1 (SAdV-1) in this study. A PCR product of the plasmid backbone was combined with SAdV-1 genomic DNA to construct an infectious clone, plasmid pKSAV1, by Gibson assembly. Restriction-assembly was performed repeatedly in the steps of intermediate plasmid isolation, modification, and restoration. The generated adenoviral plasmid was linearized by restriction enzyme digestion and transfected into packaging 293 cells to rescue E3-deleted replication-competent SAdV1XE3-CGA virus. Interestingly, SAdV1XE3-CGA could propagate in human chronic myelogenous leukemia K562 cells. The E1 region was similarly modified to generate E1/E3-deleted replication-defective virus SAdV1-EG. SAdV1-EG had a moderate gene transfer ability to adherent mammalian cells, and it could efficiently transduce suspension cells when compared with the human adenovirus 5 control vector. Restriction-assembly is easy to use and can be performed without special experimental materials and instruments. It is highly effective with verifiable outcomes at each step. More importantly, restriction-assembly makes the established vector system modifiable, upgradable and under sustainable development, and it can serve as the instructive method or strategy for the synthetic biology of adenoviruses. [ABSTRACT FROM AUTHOR]

Periodontitis is an infectious disorder that leads to irreversible loss of the surrounding attachment and bone destruction. Genetic polymorphism of cytokines has been suggested to play a role in periodontitis. This case-control study aimed to investigate the relationship between periodontitis and two single nucleotide polymorphisms (SNPs): rs1800629 (-308 G/A) and rs1799964 (-1031 T/C), in the TNF-α gene promoter area. Peripheral blood was used to prepare genomic DNA from 60 subjects with stage II to stage III periodontitis, as along with 65 control subjects. Polymerase chain reaction and restriction endonuclease digestion were used to genotype TNF-α SNPs. The distribution of both genotypes and alleles of TNF-α (-308 G/A) polymorphism did not vary between periodontitis subjects and the controls (P > 0.05). However, the CT genotype and C allele of the TNF-α (-1031 T/C) polymorphism were observed more frequently in the periodontitis subjects than in the controls, while the TT genotype and the T allele were more predominant in the control subjects than in the periodontitis patients (OR: 3.149; 95% CI: 1.494–6.639; P = 0.002 and OR: 2.933; 95% CI: 1.413–6.090; P = 0.003, sequentially). The TNF-α (-308 G/A) polymorphism potentially has no correlation with periodontitis susceptibility, whereas the TNF-α (-1031) CT genotype and C allele might be related to periodontitis among Saudi subjects. [ABSTRACT FROM AUTHOR]

Nowadays, novel Biochemistry lab techniques are being introduced at a very fast pace in scientific research. This requires development of new labs for undergraduate Biochemistry courses to equip the students with up‐to‐date techniques. However, the time limit of Biochemistry labs for undergraduate students represents a major obstacle. This article presents a clear set of laboratory exercises designed to introduce students to the use of polymerase chain reaction‐restriction‐fragment length polymorphism (PCR‐RFLP) as a means of detection of genetic variants. Three consecutive lab experiments have been designed for the undergraduate students to serve this purpose. The first session was performed in a computer lab (dry lab) where students were taught how to obtain a specific gene sequence, identify an exact single nucleotide polymorphism location, choose the target sequence for amplification, design specific primers for this particular sequence and choose the most suitable restriction enzyme from web tools. The second and third lab sessions were performed as wet labs where in the second lab session, students optimized PCR conditions and performed a successful PCR. The PCR products were kept for use in the third lab session where they utilized the selected restriction enzyme and carried out gel electrophoresis to determine the exact genotype. [ABSTRACT FROM AUTHOR]

Golden Gate cloning enables the modular assembly of DNA parts into desired synthetic genetic constructs. The "one-pot" nature of Golden Gate reactions makes them particularly amenable to high-throughput automation, facilitating the generation of thousands of constructs in a massively parallel manner. One potential bottleneck in this process is the design of these constructs. There are multiple parameters that must be considered during the design of an assembly process, and the final design should also be checked and verified before implementation. Doing this by hand for large numbers of constructs is neither practical nor feasible and increases the likelihood of introducing potentially costly errors. In this chapter we describe a design workflow that utilizes bespoke computational tools to automate the key phases of the construct design process and perform sequence editing in batches., (© 2025. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Lentinula edodes is one of the most widely consumed edible mushrooms in Korea. Mating in L. edodes is regulated by a tetrapolar system, and two unlinked genetic loci, A and B, are known to be major determinants of the mating types, as reported in other heterothallic basidiomycetes. The A locus of L. edodes encodes a pair of homeodomain (HD) transcription factors. The highly variable N-termini of these HD transcription factors contribute to the diversity among the A mating types. In this study, we developed a cleaved amplified polymorphic sequence (CAPS) marker to discriminate 11 different A mating type alleles predominant among both cultivated and wild strains. Amplification of the variable region of the A locus followed by digestion with HaeIII and EcoRI restriction enzymes enabled successful discrimination among the 11 A mating type alleles. We also evaluated the applicability of this method in the identification of two A mating types of a dikaryotic strain.

MC4R is a gene that has potential effects on growth traits such as body weight and feed intake. The usage of single nucleotide polymorphism (SNPs) in melanocortin-4 receptor (MC4R) as selection markers could help achieve effectiveness in the breeding program. This study aimed to analyze the restriction mapping based on SNPs in the MC4R gene for Bali cattle (Bos sondaicus) compared to various breeds of cattle. Partial MC4R gene was amplified using a primer (F: 5’-ACC AAT GTC AGT GAG TCC CC- 3’ and R: 5’-CTT CAT GTT GGC GCC CTG-3’) with a polymerase chain reaction (PCR) method. Genotype and allele frequencies were calculated using Chi-Square test and analyzed with Hardy–Weinberg law. Restriction enzyme was analyzed using Nebcutter V.2 to see the association between SNPs and the recognition site of restriction enzyme. The result showed four SNPs g.554 T>C, g.634 G>T, g.673 C>T, and g.742 G>A were found in the exon region. SNP g.742 G>A was found as a heterozygote genotype and the rest are SNP g.554 T>C, g.634 G>T, and g.673 C>T were found as homozygote genotypes. All SNPs were synonymous which did not change the amino acid translated. Three restriction enzymes were identified as MmeI, TspRI, and BsrI which attach to SNPs g.554 T>C, g.634 G>T, and g. 742 G>A respectively. SNPs found notably g. 742 G>A can be used as genetic markers associated with growth traits for further research on Bali cattle.

The restriction enzymes of bacteria are used to cut and insert the two strands of double-stranded DNA at different positions that cause overhangs of single stranded DNA. We developed an encoding transition table of an intuitionistic fuzzy Turing machine that processes series of restriction(cut) and ligation of DNA fragments or oligonucleotides that can be used to find the Body Mass Index of human being [ABSTRACT FROM AUTHOR]

Hühnerhirse (Echinochloa crus-galli) gewinnt als Ungras im Maisanbau zunehmend an Bedeutung. Einseitiger Einsatz von ALS-Inhibitoren erzeugt einen Selektionsdruck, der zu vermehrtem Auftreten von Resistenzen führt. Häufig handelt es sich um Target-Site-Resistenzen (TSR) mit Punktmutationen an den Positionen Ala-122, Pro- 197 oder Trp-574 des ALS-Gens. Dieses liegt in der hexaploiden Hühnerhirse in drei homologen Varianten vor. Gängige PCR-Verfahren ermöglichen nur eine gemeinsame Analyse aller Genvarianten, wodurch ein großer Teil des Informationsgehalts verloren geht. Für eine fundierte Aussage müssen die Genvarianten getrennt analysiert werden. Ein methodischer Ansatz war die allel-spezifische PCR. Hierfür wurden DNA-Polymorphismen zwischen den drei ALS-Genvarianten genutzt, um diese separat bei Position 574 hochspezifisch zu amplifizieren und zu sequenzieren. Die schwierige Struktur und geringe Polymorphismen verhinderten den Einsatz der allelspezifischen PCR an den Positionen 122 und 197. Hier wurden die drei Genvarianten gemeinsam amplifiziert und anschließend in einem mehrstufigen Ansatz mit Restriktionsenzymen spezifisch verdaut. Nach Separation, Aufreinigung und Reamplifikation der Fragmente wurden die einzelnen Genvarianten durch Pyrosequenzierung analysiert. So konnten an Position Ala-122 in allen ALS-Homologen heterozygote Mutationen nachgewiesen werden, bei Pro-197 und Trp-574 dagegen heterozygote bzw. homozygote Mutationen nur bei ALS3. Durch die Etablierung dieser Methoden können nun Resistenzmuster in Populationen von Echinochloa crus-galli detaillierter untersucht und wichtige Daten zu deren Ausbreitungsdynamik gewonnen werden.

We previously identified sequence-dependent allele-specific methylation (sd-ASM) in adult human peripheral blood leukocytes, in which ASM occurs in cis depending on adjacent polymorphic sequences. A number of groups have identified sd-ASM sites in the human and mouse genomes, illustrating the prevalence of sd-ASM in mammalian genomes. In addition, sd-ASM can lead to sequence-dependent allele-specific expression of neighbouring genes. Imprinted genes also often exhibit parent-of-origin–dependent allele-specific methylation (pd-ASM), which causes parent-of-origin–dependent allele-specific expression. However, whether most of the already known sd-ASM and pd-ASM sites are methylated or hydroxymethylated remains unclear due to technical restrictions. Accordingly, a novel method that enables examination of allelic methylation and hydroxymethylation status and also overcomes the drawbacks of conventional methods is needed. Such a method could also be used to elucidate the mechanisms underlying polymorphism-associated inter-individual differences in disease susceptibility and the mechanism of genomic imprinting. Here, we developed a simple method to determine allelic hydroxymethylation status and identified novel sequence- and parent-of-origin–dependent allele-specific hydroxymethylation sites. Correlation analyses of TF binding sequences and methylation or hydroxymethylation between three mouse strains revealed the involvement of Pax5 in strain-specific methylation and hydroxymethylation in exon 7 of Pdgfrb.

A common exercise given to students early in a molecular biology course is the creation of a restriction map of a plasmid "digested" by two restriction enzymes (RE). Meanwhile, students have learned from an early age about the properties and analyses of circles in their mathematics courses. But it is rare for students to learn using puzzle-based assignments at the intersection of molecular biology and mathematics. Therefore, we should present students with a puzzle that allows them to combine knowledge and skills from these seemingly disconnected disciplines. Here, we present a method for analyzing RE digests of circular plasmids using basic geometric principles., (© 2024 The Author(s). Biochemistry and Molecular Biology Education published by Wiley Periodicals LLC on behalf of International Union of Biochemistry and Molecular Biology.)

• Designed the RESCOT software for restriction enzyme (RE) usage in ribose-seq. • Defined and calculated the rNMP-captured region for reference genomes. • Optimized the RE usage for ribose-seq library preparation. • Validated the high rNMP-capture performance of optimized RE sets and combinations. • Expanded RESCOT usage to all existing rNMP-capture techniques. The incorporation of ribonucleoside monophosphates (rNMPs) in genomic DNA is a frequent phenomenon in many species, often associated with genome instability and disease. The ribose-seq technique is one of a few techniques designed to capture and map rNMPs embedded in genomic DNA. The first step of ribose-seq is restriction enzyme (RE) fragmentation, which cuts the genome into smaller fragments for subsequent rNMP capture. The RE selection chosen for genomic DNA fragmentation in the first step of the rNMP-capture techniques determines the genomic regions in which the rNMPs can be captured. Here, we designed a computational method, Restriction Enzyme Set and Combination Optimization Tools (RESCOT), to calculate the genomic coverage of rNMP-captured regions for a given RE set and to optimize the RE set to significantly increase the rNMP-captured-region coverage. Analyses of ribose-seq libraries for which the RESCOT tools were applied reveal that many rNMPs were captured in the expected genomic regions. Since different rNMP-mapping techniques utilize RE fragmentation and purification steps based on size-selection of the DNA fragments in the protocol, we discuss the possible usage of RESCOT for other rNMP-mapping techniques. In summary, RESCOT generates optimized RE sets for the fragmentation step of many rNMP capture techniques to maximize rNMP capture rate and thus enable researchers to better study characteristics of rNMP incorporation. [ABSTRACT FROM AUTHOR]