Restriction Enzyme Research Articles

The impact of CTLA-4 gene polymorphism on the content of organ-specific antibodies in patients with autoimmune thyroiditis among the population of Azerbaijan

BACKGROUND: The risk of developing immune tolerance to self-antigens in autoimmune thyroiditis is largely associated with a mutation in the CTLA-4 gene, the product of which negatively regulates T-cell activity. AIM: To study the A49G (rs231775) CTLA-4 polymorphism in patients with autoimmune thyroiditis in a sample of Azerbaijani residents and the effect of the frequency of identified alleles and genotypes on the level of antibodies to thyroid peroxidase and thyroglobulin. MATERIAL AND METHODS: A study was conducted in 2021–2023 involving 170 patients with autoimmune thyroiditis and 65 individuals without thyroid pathology or other autoimmune diseases, who formed a comparison group. The groups were matched by gender and age. Based on the patient history, thyroid status study results, thyroid ultrasound data, and organ-specific antibody assessment, the patients were diagnosed with autoimmune thyroiditis. The routine thyroid status study results in the comparison group were within normal limits. Genotyping of the A49G (rs231775) polymorphism was performed using polymerase chain reaction followed by digestion of the reaction product with PspEI restriction endonuclease. The results of the quantitative studies were presented as the median (Me) and interquartile range (25th quartile; 75th quartile). The correspondence of genotype frequencies to the Hardy–Weinberg equilibrium was assessed using the χ2 criterion. RESULTS: The results revealed a statistically significant increase in the frequency of the G allele (48%) compared to the control group [33.8%; p=0.039, χ2=4.27, odds ratio (OR) 1.865, 95% confidence interval (CI) 1.028–3.382] and a decrease in the frequency of the A allele (51.2%) relative to the control group (66.1%; p=0.0389, χ2=4.27, OR=0.536, CI=0.296–0.973). In 22.4% of patients with the GG genotype (p=0.005, χ2=7.86, OR=0.237, 95% CI=0.088–0.635) and 55.6% of patients with the G allele (p=0.0012, χ2=10.43, OR=0.360, 95%, CI=0.192–0.674), thyroglobulin antibody titers were more than 100 IU/ml. Thyroid peroxidase antibody levels of 100 IU/ml or more were recorded in 22.7% of study participants with the GG genotype (p=0.030) and 50.0% with the G allele (p=0.048). CONCLUSION: Elevated titers of antibodies to thyroid peroxidase and thyroglobulin were detected in patients from the population of Azerbaijan with the G allele and homozygous genotype GG of the +49A/G polymorphism of the CTLA-4 gene.

Molecular analysis of vector-borne pathogens in Eurasian badgers (Meles meles) from continental Europe

BackgroundVector-borne pathogens (VBPs) are increasing in significance in veterinary medicine and public health settings, with wildlife playing a potentially crucial role in their transmission. Eurasian badgers (Meles meles) are widely distributed across Europe. However, information currently available on the prevalence of VBPs in badgers is limited. The objective of the current study was to investigate the occurrence of Anaplasmataceae, Bartonella spp., Mycoplasma spp., Rickettsia spp., Piroplasmida, Trypanosomatida and Filarioidea in badgers and subsequently, based on the results, assess the potential risk to domestic animals, other wildlife and humans.MethodsBetween 2017 and 2021, blood or spleen samples from 220 badgers were collected in nine continental European countries: Austria (n = 7), Bosnia and Herzegovina (n = 2), Croatia (n = 22), France (n = 44), Germany (n = 16), Hungary (n = 7), Italy (n = 16), Romania (n = 80) and Serbia (n = 26). VBPs were identified by performing PCR analysis on the samples, followed by Sanger sequencing. Additionally, to distinguish between different Babesia lineages we performed restriction fragment length polymorphism (RFLP) analysis on piroplasm-positive samples, using HinfI as restriction enzyme. A phylogenetic analysis was performed on Mycoplasma spp.ResultsThe pathogens identified were Babesia sp. badger type A (54%), B (23%), and C (37%); Trypanosoma pestanai (56%); Mycoplasma sp. (34%); Candidatus Mycoplasma haematomelis (8%); Candidatus Mycoplasma haematominutum (0.5%); and Ehrlichia spp. (2%). Rickettsia spp., Bartonella spp. and filarioid nematodes were not detected among the tested samples.ConclusionsThe large sample size and diverse study populations in this study provide valuable insights into the distribution and epidemiology of the analyzed pathogens. Some of the VBPs identified in our study show high similarity to those found in domestic animals, such as dogs. This finding suggests that badgers, as potential reservoirs for these pathogens, may pose a threat not only to other wildlife but also to domestic animals in close vicinity. Continuous surveillance is essential to monitor VBPs in wildlife as a means to enable the assessment of their impact on other wildlife species, domestic animals and human health.Graphical

Cleaved Amplified Polymorphic Sequence Markers in Horticultural Crops: Current Status and Future Perspectives

DNA markers have broad applications, including marker-assisted selection (MAS) for breeding new cultivars. Currently, single nucleotide polymorphisms (SNPs) have become a preferred choice of markers for molecular geneticists and breeders. They offer many advantages, such as high abundance and coverage in the genome, codominant inheritance, locus specificity, and flexibility for high-throughput genotyping/detection formats, and they are relatively inexpensive. The availability of reference genome sequences enables precise identification of candidate genes and SNPs associated with a trait of interest through quantitative trait loci (QTL) mapping and genome-wide association studies (GWAS). Such SNPs can be converted into markers for their application in MAS in crop breeding programs. Cleaved amplified polymorphic sequence (CAPS) markers amplify short genomic sequences around the polymorphic endonuclease restriction site. This review provides insight into the recent advancements made in the development and application of CAPS markers in several horticultural plants. We discussed many new tools that aid faster and more accurate design of CAPS markers from the whole genome resequencing (WGRS) data. The developed CAPS markers offer immense application in germplasm screening and field trials, genomic loci mapping, identifying candidate genes, and MAS of important horticultural traits such as disease resistance, fruit quality and morphology, and genetic purity.

Aptamer-based sensitive fluorescence β-lactoglobulin food allergen bioassay via dual and cyclic bidirectional strand displacement amplifications.

β-Lactoglobulin (β-Lg) is a prevalent allergenic protein found in most dairy products, which poses great food safety risks for individuals with allergies, especially for infants. Sensitive and effective detection methods for such allergens are essential to reduce the risk of allergies in daily life. Herein, a fluorescent aptamer bioassay based on a dual and cyclic bidirectional strand displacement means is developed for thesensitive detection of β-Lg in infant rice porridge and milk. The aptamer in the duplex DNA probe binds β-Lg to release the assistance strand to further hybridize with two hairpins, which triggers the initiation of two cyclic amplification cycles through the polymerization, displacement, and nicking of the strands under the action of DNA polymerase and endonuclease restriction enzymes. The amplification cycles lead to the unfolding of many fluorescently quenched signal probes to exhibit substantially enhanced fluorescence recovery for detecting β-Lg. The assay can realize detection of β-Lg in concentrations as low as 4.41pM within the range of 0.01 to 10nM. Furthermore, our sensing method has the capability to discriminate β-Lg from other proteins with high selectivity, resulting in low levels of β-Lg detection in rice porridge and milk samples, demonstrating promising potentials of the developed sensing method for monitoring various food allergens.

Influence of metalloproteinase-3 (-1171 5A>6A) polymorphism on periportal fibrosis in patients with schistosomiasis mansoni, Pernambuco, Brazil

In schistosomiasis mansoni (SM), periportal fibrosis (PPF) arises due to an inflammatory response exacerbated by parasite eggs in the intrahepatic portal space, culminating in the deposition of collagen and extracellular matrix proteins. This fibrosis results from a remodeling process of the extracellular matrix, in which metalloproteinases play a significant role. The study evaluated the association between MMP-3 polymorphism (-1171 5A>6A) (rs 3025058) and sociodemographic factors with PPF in individuals with SM. This is an analytical cross-sectional study involving 242 individuals infected with S. mansoni, of these 122 were diagnosed with hepatosplenic form (HS) and 119 hepatointestinal form (HI), all from the state of Pernambuco, Brazil. Polymerase chain reaction with restriction enzyme digestion (Psyl) was used to determine the MMP-3 polymorphism (-1171 5A>6A). There was a significant association between the male gender and the HS form (OR = 1.7623 95% CI [1.0481-2.9631]; p-value = 0.0439) as well as individuals aged over 41, also had a greater chance of developing this clinical form of the disease (OR = 2.8299; 95% CI [1.5211-5.2650]; p-value = 0.0014), with greater emphasis on individuals over 61 years old (OR= 8.5541; 95% CI [3.6895-19.8326], p-value= 0.0000). There was no statistically significant association between the MMP-3 polymorphism (-1171 5A>6A) between the clinical groups (5A6A CI [0.7144-1.9879] p-value 0.5882 5A5A CI [0.0912-2.9231] p-value 0.7331 5A6A / 5A5A CI [0.6904-1.8937] p-value 0.6949). In conclusion, the results showed no association between the MMP-3 polymorphism (-1171 5A>6A) and the development of PPF. In addition, males, and age over 41 were predictive factors for the HE form of the disease in this Brazilian population.

A simple, cost-effective, and efficient method for screening CRISPR/Cas9 mutants in plants

The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9)-mediated genome editing system is widely used for targeted mutagenesis in a growing number of plant species. To streamline the screening process for mutants, especially those generated from low-efficiency editing events, there is a need for a rapid, cost-effective, and efficient method. Although several screening methods have been developed to process initial samples, these methods often tend to be time-consuming, expensive, or inefficient when dealing with larger sample sizes. Here we describe a simple, rapid, low-cost, and sensitive screening method for screening CRISPR/Cas9 mutants called PCR-Bsl I-associated analysis (PCR-BAA). This method requires only standard PCR and Bsl I restriction enzyme digestion, as well as agarose gel electrophoresis analysis. This method is particularly well suited for the efficient screening of mutants from larger populations of transformants. The simplicity, low cost, and high sensitivity of the PCR-BAA method make it particularly suitable for rapid screening of CRISPR/Cas9-induced mutants, especially those from low-efficiency editing events.

Evaluation of the Cytotoxicity Effects of Bacteriophage VbɸPA-1 on the Virulent Pseudomonas aeruginosa, A375 Human Skin Melanoma Epithelial, and HFSF PI3 Human Skin Fibroblast Cells

Background: Gram-negative bacteria are the most common infectious bacteria and are life-threatening in burn patients. Phage therapy studies can help the hospital community eliminate these bacterial pathogens. Objectives: This study aimed to evaluate the effects of the isolated bacteriophages from hospital wastewater against Gram-negative pathogenic bacteria and investigate their cytotoxicity on two human skin cell lines. Methods: The bacterial strains were isolated from burn wound infections. Bacteriophages were isolated from the hospital wastewater treatment plant in Isfahan, Iran. Transmission electron microscopy, spot tests, and restriction enzyme digestion were considered for phage identification. Finally, the lactate dehydrogenase (LDH) and tetrazolium-based MTT assays were used to realize the cytotoxicity impacts of the isolated phages. The statistical analysis of cell viability was performed using a two-way analysis of variance (ANOVA). Results: The results showed that among the isolated strains, P. aeruginosa strain NEG_RA1300 (GenBank accession number: MW845642) was sensitive to the isolated phage VbɸPA-1. The TEM results showed a possible viral taxonomy of VbɸPA-1 based on its morphology, which belonged to the Myoviridae (T4-like phages). The MTT and LDH experiments showed no cytotoxicity of VbɸPA-1 on two tested cell lines. Conclusions: Based on the results, the inhibitory effect of isolated phage on P. aeruginosa isolated from burn wounds with no toxic effect on cell lines was very significant. This phage can also be an acceptable selection against burn wound infections.

Association of polymorphism of apolipoprotein e gene with acute myocardial infarction and its short term clinical outcome

Abstract Background/Introduction Myocardial infarction (MI) is a multifactorial disease influenced by environmental and genetic factors. Apolipoprotein E (ApoE) is a protein incorporated into serum lipoproteins directing their catabolism via binding to receptors. It is a structural component of both chylomicrons and very low density lipoprotein (VLDL) remnants. ApoE is thought to play a central role in atherosclerosis by participating in overall plasma cholesterol homeostasis. In addition to other classical risk factors, positive family history of cardiovascular disease (CVD) has been found to be significantly and independently associated with MI in epidemiological studies (1). Besides traditional cardiovascular risk factors it is likely that additional genetic factors like ApoE gene polymorphism may interact with environmental factors to determine overall cardiovascular risk of an individual to develop MI. Purpose The purpose of our study was to determine the risk of association of variants of ApoE gene with Acute Myocardial Infarction. Moreover, we also wanted to study the impact of ApoE gene polymorphism in short term major adverse cardiac events (MACE) in patients with MI not willing for coronary intervention. Methods In this case control study a total of 200 consecutively admitted AMI patients admitted to the coronary care unit of our hospital were enrolled to the study along with 200 age and gender matched controls admitted during the same time period . ApoE gene polymorphism was detected by DNA extraction, DNA amplification by PCR in a DNA thermal cycler followed by restriction enzyme digestion(2). Chi square test was used to investigate the difference in genotype and allele frequencies in casend controls and also between observed and expected frequencies in the realm of Hardy-Weinberg equilibrium. Odds Ratio (OR) with 95 % confidence interval and p values were calculated while analysing the association of different genotypes of ApoE genetic polymorphism with the disease and MACE. Logistic regression analysis was carried out amongst the groups under consideration to find the unadjusted OR and adjusted OR of the genotypes in relation to the disease.p<0.05 was considered as significant Analysis was done in SPSS software. Results Besides the traditional risk factors Apo E4 allele (OR 2.298, CI 1.305-4.044 ) and Apo E3/E4 genotype (OR 2.116, CI 1.008-4.443, p=0.048) showed strong of association with the disease. There was a higher population of Apo E3/E4 genotypes and Apo E2/E3 genotypes with MACE events (25.6% and 28.6%), while the common ApoE3/E3 genotypes had a MACE event of 10.5 %. However, the difference was not significant (p=0.071, Χ 2 =8.63 df =4). Similarly, ApoE4 allele showed a non significantly higher MACE events (p=0.22, Χ 2 =3.01 df =4) Conclusion ApoE4 allele and ApoE3/E4 genotype are strongly and independently associated with AMI.RISK FACTORS AND RISK OF MIGenotype and MACE

PcdA promotes orthogonal division plane selection in Staphylococcus aureus.

The bacterial pathogen, Staphylococcus aureus, grows by dividing in two alternating orthogonal planes. How these cell division planes are positioned correctly is not known. Here we used chemical genetic screening to identify PcdA as a division plane placement factor. Molecular biology and imaging approaches revealed non-orthogonal division plane selection for pcdA mutant bacteria. PcdA is a structurally and functionally altered member of the McrB AAA+ NTPase family, which are often found as restriction enzyme subunits. PcdA interacts with the tubulin-like divisome component, FtsZ, and the structural protein, DivIVA; it also localizes to future cell division sites. PcdA multimerization, localization and function are NTPase activity-dependent. We propose that the DivIVA/PcdA complex recruits unpolymerized FtsZ to assemble along the proper cell division plane. Although pcdA deletion did not affect S. aureus growth in several laboratory conditions, its clustered growth pattern was disrupted, sensitivity to cell-wall-targeting antibiotics increased and virulence in mice decreased. We propose that the characteristic clustered growth pattern of S. aureus, which emerges from dividing in alternating orthogonal division planes, might protect the bacterium from host defences.

Golden EGG, a simplified Golden Gate cloning system to assemble multiple fragments

The Golden Gate method is an efficient tool for seamless assembly of multiple DNA fragments, which uses Type IIS restriction endonucleases, cleaving the DNA outside of their recognition site to release DNA parts from PCR fragments or entry clones, thus allowing the design of overhangs for ligation at will. However, the construction of the entry clones requires the use of other restriction enzyme(s) or cloning techniques and different entry vectors for the individual overhangs. Here, we present a simplified Golden Gate cloning approach termed Golden EGG. It features (1) a single entry vector with a specific cloning site to host the DNA parts; (2) a unique primer design to create the restriction enzyme recognition site to release the fragments with the overhangs at will; (3) the use of a single Type IIS enzyme for the construction of both the entry and destination clones; (4) a specific temperature profile during the digestion-ligation reaction. Our user-friendly, streamlined method retains the key attributes of the Golden Gate technique, while offering the potential to generate compatible parts with any existing Golden Gate toolkit and to be accessible to a wide user base without the need for extensive acquisition of new vectors or expensive enzymes.

Dual quenching ECL strategy based on AgInZnS quantum dots and a new co-reaction promoter oxygen vacancy-modified P5AIn/TiO2 for sensitive CEA detection

A novel electrochemiluminescence (ECL) biosensor for the sensitive detection of carcinoembryonic antigen (CEA) was constructed, utilizing environmentally friendly AgInZnS quantum dots (AgInZnS QDs) and a new co-reaction promoter enriched with oxygen vacancies, namely titanium dioxide/poly(5-aminoindole) (Ov-TiO2/P5AIn). The composite of P5AIn and TiO2 significantly enhanced the reaction rate of AgInZnS QDs with tripropylamine (TPrA). In addition, the presence of oxygen vacancies significantly enhances the electron migration rate of the electrode material, leading to more efficient catalysis of TPrA+ generation and a significant enhancement of ECL signals. Furthermore, a dual-quenching sensing strategy was employed using cuprous oxide-triaminophenol (Cu2O@APF) as the quencher, enabling the biosensor to switch. Through dual quenching of the ECL emission from AgInZnS QDs by Cu2O and APF, the biosensor achieves an extremely low “off” state of the ECL signal. Furthermore, a signal amplification strategy driven by the synergistic action of exonuclease III (ExoIII) and restriction endonuclease (Nt.BbvCI) was designed for a dual-site DNA walker, achieving cyclic amplification of the target signal and ECL signal, thereby turning the signal “on” again. The above mechanism significantly improved the specificity and sensitivity of the biosensor for CEA detection. The prepared biosensor exhibits good stability, selectivity, and sensitivity. The linear range for CEA detection is 5 × 10−14 to 10−8 M, with a detection limit of 16 fM. Additionally, the biosensor demonstrates satisfactory analytical capability for CEA in real blood samples. This strategy provides a reliable new approach for the sensitive detection of CEA and other tumor markers.

Genotype of Varicella-zoster virus isolated in Jiangsu, China

ObjectiveTo analyze the genotypes of VZV in Jiangsu province to identify vaccine strains and wild strains, providing a molecular biological background for the effective prevention and control of varicella.MethodStratified sampling was used to collect herpes fluid or throat swab from patients diagnosed with varicella. ORF22 was carried out, and the restriction enzyme site of ORF38, ORF54 and ORF62 were detected.ResultsAll 207 virus strains were Clade 2 type by sequencing the PCR products of ORF22. The sequencing results showed that five SNP sites changed compared to the Dumas reference strain(Clade 1). From A to G at 37,902, from T to c at 38,055, from A to C at 38,081, and from G to A at 38,177, from G to A at 39,394. The prevalent VZV genotypes in Jiangsu is consistent with the P-Oka. The restriction enzyme site analysis of PCR amplification products from ORF38 (PstI), ORF54 (BglI), ORF62 (SmaI) showed that all 207 virus strains were wild-type. There were two different types of the wild strains, and 183 strains (88.4%) were PstI (+), BglI (+), SmaI (-). The wild strains between different regions showed no significant differences (χ2 = 0.05, P = 0.982).ConclusionsThe prevalent VZV genotypes are Clade 2 and the prevalent virus strains are wild strains in Jiangsu Province, the primary wild strain observed is mainly PstI (+), BglI (+), SmaI (-).

Association of STAT1 gene with milk fat and protein yield in Holstein Friesian crossbred cattle maintained in the sub-tropical climate of India.

Signal transducers and activators of transcription (STAT) genes are involved in signal mediation of various hormones and cytokines. STAT1 located on chromosome number 2 is involved in mammary gland development and is associated with milk composition traits in bovines. This study aimed to find any relationship and impact of STAT1/BspHI gene with milk fat and protein yields in a herd of Holstein Friesian (HF) crossbred cattle of sub-tropical climate of Northern India. Milk composition data of 535 adult HF crossbred cows for a period of 12 years was collected from the records maintained at Livestock Farm, Guru Angad Dev Veterinary and Animal Sciences University. First lactation data of 222 animals was chosen for further analysis. After data correction for non-genetic factors (season of calving, period of calving, interaction effect of season and period of calving and age at first calving) these animals were categorised into two groups based on corrected high and low milk fat and protein yields. Forty animals were then selected for blood collection and further laboratory analysis. Amplified using PCR-RFLP technique, the 314 bp STAT1 gene was digested using BspHI restriction enzyme. C-T polymorphism at nucleotide position 201 and 260 of the STAT1 amplicon was observed. At 201, for genotype AA and Aa, the genotypic frequencies were 0.80 and 0.20%. At 260, for genotype BB and Bb, the genotypic frequencies were 0.25 and 0.75%. Least square analysis showed a significant association of all genotypes with milk fat and protein yields. Hence, STAT1 can be used as a potential candidate gene to aid in better animal selection in breeding programmes.

Optimising Transformation Efficiency in Borrelia: Unravelling the Role of the Restriction-Modification System of Borrelia afzelii and Borrelia garinii

Borrelia spp. are transmitted to humans by the bite of an infected tick. In Europe, Borrelia afzelii and Borrelia garinii are the main causative agents of Lyme borreliosis, one of the most prevalent tick-borne diseases in the northern hemisphere. In bacteria such as Borrelia spp., a restriction-modification system (RMS) protects against the harmful introduction of foreign DNA. The RMS comprises two activities: methyltransferase and endonuclease. This study is aimed to characterize the RMS of B. afzelii and B. garinii. First, we identified potential RMS genes. The predicted genes were cloned into a methylase-deficient Escherichia coli strain and digested with methylation-sensitive restriction enzymes to verify methyltransferase activity. Additionally, the RMS proteins were purified to evaluate endonuclease activity. Subsequently, methylated and unmethylated plasmids were used to investigate the effect of methylation on endonuclease activity and transformation efficiency. We identified four possible RMS genes in B. afzelii and four RMS genes in B. garinii. We analyzed the presence of these genes in patient isolates and observed a high degree of heterogeneity. The restriction pattern of DNA methylated by each of the four recombinantly expressed genes provided strong evidence that all encode adenine-specific methyltransferases. After 24 h of incubation with purified RMS proteins, we observed complete digestion of unmethylated plasmid DNA, demonstrating endonuclease activity. Finally, we proved that methylation protects against endonuclease activity and increases transformation efficiency.

Assessment of the Degree Of DNA Methylation in Lymphocytes аfter а Single Blood Irradiation in vitro

DNA methylation is one of the processes of epigenetic regulation of the genome, which is sensitive to the influence of endogenous and exogenous factors. The effect of ionizing radiation on the genome is accompanied by a change in the degree of DNA methylation, which can be dose-dependent and persist for a long time after radiation exposure. The objective of the study was to assess the degree of DNA methylation of blood lymphocytes after a single exposure to gamma radiation at a dose of 1.5 Gy using wide-genome bisulfite sequencing. The study included 10 conditionally healthy male employees of the ionizing radiation facility who were not exposed to radiation and did not suffer from chronic diseases. The material was whole blood: 0 Gy (control samples) and 1.5 Gy (experimental samples irradiated with gamma radiation). After irradiation with subsequent cultivation of whole blood, DNA isolation and bisulfite sequencing of limited sets of genomic loci (Reduced representation bisulfite sequencing) was performed using XmaI restriction enzyme (XmaI-RRBS). 41 genes were identified, including 26 genes (HOXD4, PADI2, FOXK1, FTCD, PRDM16, TOM1, PPP1R14A, FLNB, OR1F1, RARA, CRTAC1, AP5B1, ARL5C, NOC2L, MAMDC4, FGFRL1, PPFIA3, CUX2, ANKRD20A19P, FAM83H-AS1, CBFA2T3, POLN, MIR4458HG, FNBP1, SPIRE2, and ZSCAN10) have a tendency to hypomethylation DNA, and another 15 genes (CHRNA4, SEPTIN9, ZNF174, ELK3, NFAM1, ALG10, SOX8, KLHL30, URI1, HBZ, KLF14, MYO16, MYEOV, DMKN, and PAX7) tend to have hypermethylated state detected in at least 50٪ of the experimental samples. Thus, the genes identified in this study can be promising markers of radiation exposure and, in the future, be used to develop a new type of biological dosimetry – epigenomic dosimetry of personnel in contact with ionizing radiation sources in the course of their professional activities.

Converting Multiple- to Single-DNA-Tethered Beads and Removing Only-One-End-Tethered DNA in High-Throughput Stretching.

S-DNA is a double-stranded DNA that forms under tensions of >65 pN. Here, we report that S-DNA resists the cleavage of Cas12a and the restriction endonuclease SmaI. Taking advantage of this resistance, in magnetic tweezer experiments, we developed an assay to convert multiple-DNA-tethered beads into single-DNA-tethered beads and remove the only-one-end-tethered DNA molecule by cleaving the DNA that does not transition to S-DNA at about 80 pN. When multiple DNA molecules are tethered to a single bead, they share the tension, exist in the B-form, and allow the cleavage. Only-one-end-tethered DNA molecules, free of tension, are also cleaved. In versatile types of experiments, we proved the broad applications of this assay: measuring the correct DNA elasticity and DNA condensation dynamics by avoiding the false results due to interference of only-one-end-tethered DNA molecules and quantifying the accurate cleavage rates of Cas12a and the restriction endonucleases by eliminating the error caused by multiple-DNA-tethered beads. This convenient assay ensures correct and accurate results in high-throughput DNA stretching experiments.

My Early Years of Yeast Mitochondrial Genetics.

There have been massive technological advances in molecular biology and genetics over the past five decades. I have personally experienced these advances and here I reflect on those origins, from my perspective, studying yeast mitochondrial genetics leading up to deciphering the functions of the mitochondrial genome. The yeast contributions commenced in the middle of the last century with pure genetics, correlating mutants with phenotypes, in order to discover genes, just like the early explorations to discover new lands. The quest was to explore the mitochondrial genome and find its genes and their products. It was most fortunate that DNA sequencing technologies became available in the late 1970s, and laboratories were restructured enormously to keep pace with the emerging technologies. There were considerable costs in equipping laboratories, purchasing ultracentrifuges and restriction endonucleases, and undertaking DNA sequencing; additionally, workers required special safety gear.

An Investigation of Polymorphism on the FASN, SCD1, and SLC27A3 Genes in Sheep

Milk traits of sheep are affected by many environmental and genetic factors. These traits are quantitative traits and are determined by many genes. This study aimed to investigate polymorphisms of milk-related Fatty-acid synthase (FASN), Stearoyl-CoA desaturase (SCD1), and solute carrier 27A3 (SLC27A3) genes in cross-bred Hamdani sheep. Blood samples were collected from 100 healthy cross-bred Hamdani ewes from the jugular vein into K3-EDTA-containing tubes. Genomic DNA was extracted using a commercial DNA isolation kit. PCR products of FASN (275 bp), SCD1 (225 bp), SLC27A3-P1 (341 bp), and SLC27A3-P2 (319 bp) were subjected to restriction fragment length polymorphism (RFLP) analysis using SSil (AciI), Cfr13I, AluI, and Hpy188III the restriction enzyme, respectively. However, all gene regions were found to be monomorphic. In the study, only TT, AA, AA, and GG genotypes were detected for FASN, SCD1, SLC27A3-P1, and SLC27A3-P2, respectively. Allele and genotype frequencies were 1.00 for all genotypes and alleles. Although this study did not reveal favorable genotypes in FASN, SCD1, and SLC27A3 genes that can be used for milk traits, more comprehensive studies with larger sample sizes should be conducted to investigate polymorphisms in cross-bred Hamdani sheep.

Golden gate method in biology and medicine

The aim of this review was to describe and compare molecular cloning methods for assembling genetic constructs. Genetic therapy is one of the rapidly developing fields of modern medicine, so special attention in this review is paid to parameters such as speed, accuracy, and efficiency of cloning, as these are critical factors in creating gene therapy agents. Special attention is given to the Golden Gate method, which is based on the use of type IIS restriction endonucleases, as this approach simplifies the cloning process and increases its efficiency through standardized design and a minimal set of enzymes. Alongside Golden Gate, the review also discusses more innovative molecular cloning methods, Gateway and Gibson, in terms of their potential use for addressing fundamental and applied challenges in regenerative medicine.

Construction of a chemiluminescent biosensor based on enzymatic extension and click chemistry for sensitive measurement of MGMT activity in human breast tissues

O6-methylguanine methyltransferase (MGMT) is responsible for dealkylation of naturally occurring O6-methylguanines, and it is closely related with DNA replication, transcription, and cancers. Herein, we develop a chemiluminescent biosensor based on enzymatic extension and click chemistry for sensitive measurement of MGMT activity. When MGMT is present, the MGMT-catalyzed demethylation reaction initiates the cleavage of biotinylated dumbbell probes by PvuII restrictive enzyme, releasing two DNA fragments with 3′-OH end. The resultant DNA fragments can trigger terminal transferase (TdT)- and click chemistry-assisted isothermal amplification to obtain abundant G-rich sequences. The G-rich sequences can be captured by magnetic beads to produce a high chemiluminescence signal. This biosensor can greatly amplify the chemiluminescence signal, facilitating label-free and template-free measurement of MGMT. Especially, the introduction of dumbbell probe and PvuII enzyme can efficiently eliminate the false positive and improve the assay specificity. This biosensor possesses high sensitivity with a detection limit of 1.4 × 10−9 ng/μL, and it may accurately quantify the intracellular MGMT. Importantly, this biosensor can be used to screen the MGMT inhibitors and distinguish the MGMT level in breast tumor tissues and normal tissues, with great potential in drug discovery and cancer diagnosis.