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There are controversial reports on the restoration of eroded telomere length in offspring produced by somatic cell nuclear transfer (SCNT) in different animal species. To the best of our knowledge, no earlier studies report the telomere length in naturally produced or cloned animals in any of the camelid species. Therefore, the present study was conducted to estimate the telomere length in dromedary camels produced by SCNT, the donor cells, and their age-matched naturally produced counterparts by Terminal Restriction Fragment (TRF) length analysis and real-time Q PCR T/S ratio methods. Genomic DNA was extracted from venous blood collected from 6 cloned animals and their age-matched counterparts. Using the southern blot technique, digested DNA was blotted onto a positively charged nylon membrane, and its hybridization was carried out using telomere (TTAGGG)n specific, DIG-labeled hybridization probe (Roche Diagnostics, Germany) at 42 °C for 4 h. Stringent washes were carried out at the same temperature, followed by a chemiluminescence reaction. The signals were captured using the Azure Biosystems C600 gel documentation system. A TeloTool program from MATLAB software with a built-in probe intensity correction algorithm was used for TRF analysis. The experiment was replicated three times, and the data, presented as mean ± SEM, were analyzed using a two-sample t-test (MINITAB statistical software, Minitab ltd, CV3 2 TE, UK). No difference was found in the mean telomere length of cloned camels when compared to their naturally produced age-matched counterparts. However, the telomere length was more (P 0.05) than that of the somatic cells used for producing the SCNT embryos. A moderate positive Pearson correlation coefficient (r = 0.6446) was observed between the telomere lengths estimated by TRF and Q PCR T/S ratio method. In conclusion, this is the first study wherein we are reporting telomere length in naturally produced and cloned dromedary camels produced by somatic cell nuclear transfer. We found that telomere lengths in cloned camels were similar to their age-matched naturally produced counterparts, suggesting that the camel cytoplast reprograms the somatic cell nucleus and restores the telomere length to its totipotency stage.

Objectives Interferon-gamma (IFNg) is an immune-regulatory cytokine with a role in host responses to periodontitis. Genetic factors have been reported to modify the corresponding protein expression. The objective of this study was to evaluate the association and role of IFNg polymorphisms, such as IFNg +874 A/T, and the susceptibility to periodontitis. Materials and Methods A total of 100 unrelated subjects were included in the present study. Genomic deoxyribonucleic acid (DNA) was obtained from peripheral blood of 43 patients with mild periodontitis and 57 patients with severe periodontitis. The determined clinical parameters of periodontitis included probing depth, clinical attachment loss, and papilla bleeding index. The oral hygiene indicators were also assessed. The level of IFNg was determined from the gingival crevicular fluid by enzyme-linked immunosorbent assay technique. The IFNg +874 A/T polymorphisms were analyzed from peripheral blood by the method of restriction fragment length polymorphism-polymerase chain reaction. Statistical Analysis Statistical analysis of the results was conducted using chi-squared testing for categorical data. Independent t-tests and Mann–Whitney U tests were used for numeric data. Kruskal–Wallis testing was used to compare genotypes concerning for IFNg +874 A/T polymorphism. A p-value Results Analysis of the IFNg +874 A/T polymorphism showed no significant differences with the level of IFNg. No significant differences were observed either in IFNg +874 A/T polymorphism between the subjects with mild periodontitis and those with severe periodontitis (p > 0.05). The subjects with severe periodontitis showed marginally but not significantly higher levels of IFNg compared with subjects with mild periodontitis (p > 0.05). Conclusion The polymorphism of IFNg +874 A/T was not associated with the level of IFNg nor with the risk of periodontitis in this study.

Banana belonging to the family Musaceae is the fourth most important staple food crop after rice, wheat, and maize. Genomic composition and genetic variation among 25 Musaceae genotypes, collected from different regions of Manipur, were evaluated using PCR–RFLP and RAPD and ISSR markers. Restriction-site variations in the internal transcribed spacers (ITS) of the nuclear ribosomal RNA genes were used to distinguish the genotypes and their genomic constitutions. PCR amplification using specific primers produced a 700-bp fragment in all the genotypes studied. This fragment that is equivalent in size to the ITS of most plants was digested with RsaI, revealing 530-bp fragment that is unique to ‘A’ genome and 350 bp and 180-bp fragments that were unique to ‘B’ genome alone, and thus, the genotypes were differentiated as constituting of either the A or B genome alone or having both the A and B genomic compositions. In the wild and semi-wild genotypes, unique restriction fragments were observed. Genetic variations among the genotypes were determined using 15 RAPD and 15 ISSR markers. The respective percentage of polymorphisms for RAPD and ISSR markers was 99.79% and 99.70%. Similarity coefficients in RAPD analysis ranged from 0.14 to 0.95 and in ISSR analysis from 0.30 to 0.96. The polymorphism information content (PIC) scores for RAPD markers ranged from 0.20 to 0.29 and that of ISSR markers was 0.25 to 0.36. The Mantel Z-test between the two Jaccard’s similarity matrices gave r ≥ 0.93, indicating a very good fit correlation between RAPD- and ISSR-based similarities. The RAPD and ISSR analyses could identify distinctive cultivars and wild species that remained conspicuously distinct in both marker systems.

Human papillomaviruses (HPV) are known to play a central etiological role in the development of cervical cancer. General HPV genotyping methods consist of PCR with consensus primers combined with various detection methods. The aim was to develop HPV L1 DNA reference materials to evaluate the sensitivity, specificity, and accuracy of genotyping results obtained from the HPV DNA Genotyping Chip (HPV CHIP) and RFMP assays. In this study, the Ministry of Food and Drug Safety (MFDS) established reference DNA materials for the L1 gene from 41 subtypes of anogenital HPV to aid in genotyping human papillomavirus (HPV) strains. Of these, 22 subtypes were obtained from cervical scrape samples of Korean women and 19 subtypes were synthesized. These reference materials include 13 high-risk types (HPV-16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, and 68), 3 probable high-risk types (HPV-26, 53, and 66), 16 low-risk types (HPV-6, 10, 11, 27, 34, 40, 42, 43, 44, 54, 55, 61, 70, 72, 73, and 81), and 8 undetermined-risk types (HPV-3, 57, 62, 67, 69, 71, 74, and 84). After confirming the sequences by standard methods, these HPV L1 DNA reference materials were then used to compare results from the HPV DNA Genotyping Chip (HPV CHIP) and restriction fragment mass polymorphism (RFMP) assays. Data collected from the HPV CHIP and RFMP assay showed comparably high sensitivity and accuracy. Both assays could detect 102 or more copies/μl of HPV L1 DNA from 39 types of HPV, with higher accuracy in detecting samples with mixed types of HPV. The present study confirms the HPV L1 DNA reference materials developed by MFDS are reliable and useful for the evaluation of HPV genotyping assays.

Made available in DSpace on 2021-06-25T23:35:56Z (GMT). No. of bitstreams: 0 Previous issue date: 2021-01-08 Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ) Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) This study aimed to genetically characterize Toxoplasma gondii isolates obtained from free-range chickens reared in the metropolitan region of the state of Rio de Janeiro, Brazil, and to evaluate the morbidity and histological changes associated with these isolates in mice. A mouse bioassay was used to isolate T. gondii from a pool of tissue samples (brain, heart, and thigh muscles) collected from 163 chickens. The 36 isolates obtained were genetically characterized by restriction fragment polymorphism (PCR-RFLP) analysis of the SAG1, 5 '-3 ' SAG2, aSAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1, Apico, and CS3 genomic regions. Seventeen atypical genotypes were identified and nine of them were reported for the first time. All identified genotypes caused clinical signs and histological changes in mice, with the majority being associated with high cumulative morbidity (65%) and severe or very severe histological changes (76%). The exclusive identification of atypical genotypes, with a predominance of new genotypes, indicates great genetic diversity of T. gondii in the region studied. In addition, the finding that all identified genotypes caused clinical signs and often severe histological changes in mice suggests potentially relevant virulence of these strains. Fundacao Oswaldo Cruz Fiocruz, Inst Nacl Infectol Evandro Chagas, Ave Brasil 4365, Rio De Janeiro, RJ, Brazil Fundacao Oswaldo Cruz Fiocruz, Inst Oswaldo Cruz, Ave Brasil 4365, Rio De Janeiro, RJ, Brazil Univ Oeste Paulista, Fac Ciencias Agr, Presidente Prudente, SP, Brazil Univ Estadual Paulista, Fac Med Vet & Zootecnia, Botucatu, SP, Brazil Univ Fed Fluminense, Inst Biomed, Rua Prof Hernani Pires de Melo 101, Niteroi, RJ, Brazil Univ Estadual Paulista, Fac Med Vet & Zootecnia, Botucatu, SP, Brazil FAPERJ: APQ1 E-26/110.937/2009 FAPERJ: APQ1 E-26/111.977/2012 FAPERJ: TCT E-26/102.303/2011 FAPERJ: JCNE E-26/102.247/2013 CAPES: 001 CNPq: MCT/CNPq 10/2010-AT-NM

While clonal heterogeneity has been demonstrated in most cancers, quantitative assessment of individual tumor clones has not been translated to inform clinical practice. A few methods have been developed to investigate the tumor clonality of adult T cell leukemia/lymphoma (ATLL), but currently there is no clinically translatable method available for quantifying individual tumor clones in ATLL patients. Here, we present a methodology to assess the tumor clonality of ATLL and quantify patient-specific tumor clones in a clinical setting. The methodology consists of three steps: (1) selective amplification of restriction fragments containing a human T cell leukemia virus type 1 (HTLV-1) integration site, (2) amplicon deep sequencing to estimate the clonal structure and identify HTLV-1 integration sites of dominant clones, and (3) digital PCR targeting the HTLV-1 integration sites of the dominant clones to quantify specific tumor clones. We successfully tracked individual tumor clones using this approach and demonstrated that each clone had a distinct response to therapies. The procedure is straightforward and clinically feasible, which should facilitate the proper assessment and management of ATLL., Graphical Abstract, A novel procedure is presented for detecting tumor clonality and quantifying individual tumor clones in adult T cell leukemia/lymphoma, which could facilitate the monitoring of patient-specific tumor clones in clinical practice.

In the present study, a total of eight sourdough samples were collected from three different bakeries at two different times in Turkey. Also, laboratory-scale sourdough production was conducted by daily back-slopping for 7 days. Microbiological and chemical properties of the sourdoughs were investigated. Yeast species in the sourdoughs were identified by subjecting all presumptive yeast cultures to internal transcribed spacer region amplification of the 5.8S rRNA gene, restriction fragment length polymorphism analysis using Hae III, Hha I, and Hinf I endonucleases, and sequence analysis of the D1/D2 domain of the 26S rDNA gene. A total of seven profiles were determined according to the restriction fragments. Totally, 148 yeast isolates were identified at the species level (≥400 bp, 99% identity) as Saccharomyces cerevisiae (106), Kazachstania bulderi (11), Pichia fermentans (nine), Pichia membranifaciens (eight), Kazachstania servazzii (seven), Kazachstania unispora (four), and Hanseniaspora valbyensis (three). Although collected sourdoughs were produced without using baker's yeast, S. cerevisiae was the most frequently isolated yeast species. This can be related to the contamination of the bakery environment with commercial baker's yeast during the production of other bakery products. The pH and acidity levels of the collected sourdough samples ranged from 3.71 to 3.96 and 6.78 to 23.93 mL 0.1 N NaOH/10 g dough, respectively. Mean values of the content of maltose + sucrose, glucose, fructose, lactic acid, and acetic acid were 2.43, 1.57, 2.67, 7.30, and 1.40 g/kg, respectively. Due to the artisan and region-dependent handling of the sourdough, different biochemical patterns were observed among the collected samples.

The oxidative modification of low-density lipoprotein (LDL) in the arterial wall plays a pivotal role in the initiation and progression of atherosclerosis which is a complex and progressive disorder. Paraoxonase1 (PON1), which is required for lipid metabolism, is believed to protect LDL from oxidation. The relationship between PON1 gene Leusin55Methionin (L55M) and Glutamine192Arginine (Q192R) polymorphisms in western Iranians with atherosclerosis and its association with enzyme activity and oxidized low-density lipoprotein (oxLDL) were examined in the present study. In this study, blood specimens were collected from 145 healthy individuals and 154 patients with atherosclerosis proven by angiography referred to Shahid Madani Hospital, Khorramabad, Iran. Genomic deoxy ribonucleic acid (DNA) was extracted from whole blood. For all the subjects, restriction fragment length polymorphism-polymerase chain reaction (RFLP-PCR) was carried out for the detection of L55M and Q192R polymorphisms. PON1 enzyme activity and the level of oxLDL were also evaluated. There was a 3.114-fold increase in the risk of developing atherosclerosis in the subjects presenting the PON1L55M, MM genotype compared to those with the LL genotype (OR 3.114; 95% CI 1.412–6.870). PON1Q192R polymorphism in the PON1 gene was not associated with atherosclerosis. Patients with atherosclerosis had significantly higher oxLDL and reduced PON1 enzyme activity (P

Magnaporthe oryzaeis a filamentous ascomycete fungus that causes devastating diseases of crops that include rice and wheat, and a variety of turf, forage and wild grasses. Strains from ryegrasses possess highly stable chromosome ends that undergo frequent rearrangements during vegetative growth in culture andin planta. Instability is associated with the presence of two related retrotransposons (MagnaportheoryzaeTelomericRetrotransposons - MoTeRs) inserted within the telomere repeat tracts. The objective of the present study was to determine the mechanisms by which MoTeRs promote telomere instability. Targeted cloning, restriction mapping, and sequencing of both parental and novel telomeric restriction fragments, along with MinION sequencing of DNA from three single-spore cultures, allowed us to document the molecular alterations for 109 newly-formed telomeres. Rearrangement events included truncations of subterminal rDNA sequences; acquisition of MoTeR insertions by “plain” telomeres; insertion of the MAGGY retrotransposons into MoTeR arrays; expansion and contraction of subtelomeric tandem repeats; MoTeR truncations; duplication and terminalization of internal sequences; and breakage at long, interstitial telomeres generated during MoTeR insertion. Together, our data show that when MoTeRs invade the telomeres, they can dramatically perturb the integrity of chromosome ends, leading to the generation of unprotected DNA termini whose repair has the potential to generate chromosome alterations that extend well into the genome interior.

The genus Elymus L., together with wheat, rye, and barley, belongs to the tribe Triticeae. Apart from its economic value, this tribe is characterized by abundance of polyploid taxa formed in the course of remote hybridization. Single-copy nuclear genes are convenient markers for identification of source genomes incorporated into polyploids. In the present work, a CAPS-marker is developed to distinguish basic St, H, and Y genomes comprising polyploid genomes of Asiatic species of the genus Elymus. The test is based on electrophoretic analysis of restriction patterns of a PCR-amplified fragment of the gene coding for beta-amylase. There are about 50 Elymus species in Russia, and most of them are supposed to possess one of three haplome combinations, StH, StY and StHY. Boreal StH-genomic species endemic for Russia are the least studied. On the basis of nucleotide sequences from public databases, TaqI restrictase was selected, as it produced patterns of restriction fragments specific for St, H, and Y haplomes easily recognizable in agarose gel. A sample of 68 accessions belonging to 32 species was analyzed. In 15 species, the earlier known genomic constitutions were confirmed, but in E. kamoji this assay failed to reveal the presence of H genome. This unusual H genome was suggested to originate from a different Hordeum species. In 16 species, genomic constitutions were identified for the first time. Fifteen accessions from Asian Russia possessed the genomic constitution StStHH, and E. amurensis, phylogenetically close to the StY-genomic species E. ciliaris, had the genomic constitution StStYY. It is inferred that the center of species diversity of the StH-genomic group is shifted to the north as compared to the center of origin of StY-genomic species, confined to China. Key words: Elymus; taxonomy; allopolyploids; genome constitution; CAPS markers.Род Elymus L. наряду с пшеницей, рожью и ячменем принадлежит к трибе Triticeae. Помимо своего хозяйственного значения, эта триба характеризуется широким распространением аллополиплоидных таксонов, которые формируются в ходе межвидовой и межродовой гибридизации и последующих преобразований вовлеченных в гибридизацию диплоидных геномов. Для идентификации исходных геномов в составе полиплоидов удобны малокопийные ядерные гены, менее подверженные процессам реорганизации, чем повторенные некодирующие элементы. В настоящей работе разработан удобный CAPS-маркер для различения базисных геномов St, H, Y, входящих в состав азиатских видов рода Elymus, с помощью электрофоретического анализа фрагментов рестрикции ПЦР-амплифицированного участка гена, кодирующего β-амилазу. В России распространено около 50 видов Elymus предположительно трех гапломных комбинаций: StH, StY и StHY. Наименее изученными остаются бореальные StH-геномные виды – эндемики Российской Федерации. По результатам анализа ранее изученных разными авторами нуклеотидных последовательностей гена β-амилазы была отобрана эндонуклеаза рестрикции TaqI, которая имела различающиеся по положению сайты узнавания в составе вышеуказанного фрагмента из геномов St, H и Y. В результате расщепления ПЦР- продукта эндонуклеазой TaqI у каждого из исходных гапломов формировался специфичный паттерн фрагментов рестрикции, легко визуализируемый в агарозном геле. Проанализирована выборка из 68 образцов, принадлежащих 32 видам. У 15 видов была подтверждена ранее известная геномная конституция, у E. kamoji этот метод не позволил выявить присутствие генома H. Предполагается возможное происхождение данного варианта генома H от другого вида Hordeum. У 16 видов геномная конституция определена впервые. Показано, что большинство изученных видов бореальной группы видов из Сибири и Российского Дальнего Востока имеют геномную конституцию StStHH. Исключение составил E. amurensis, филогенетически близкий к StY-геномному виду E. ciliaris и также имеющий геномный состав StStYY. Сделан вывод, что основное видовое разнообразие StH-геномной группы находится севернее центра происхождения большинства StY-геномных видов рода.

Aims Systemic lupus erythematosus (SLE) is a chronic inflammatory autoimmune disease. Prostaglandins E2 (PGE2), the product of the cyclo-oxygenase 2 (COX-2) enzyme, has critical roles in the etiology of autoimmune diseases. PGE2 level is controlled by a balance between its synthesis mediator (COX-2 enzyme) and its catabolic key enzyme (15-hydroxyprostaglandin dehydrogenase [15-PGDH] enzyme). In the present study, the associations of genotypic polymorphisms in COX-2 and 15-PGDH with SLE were investigated. Methods One hundred and sixty SLE patients and 160 healthy controls participated in the study. The polymerase chain reaction - restriction fragments length polymorphism method was used for genotyping. The COX-2 rs2745557 G/A and 15-PGDH rs8752 G/A polymorphisms were investigated. Results Regarding the COX-2 rs2745557 single nucleotide polymorphism, there was no significant association between COX-2 rs2745557 polymorphism and SLE. However, the dominant models showed a marginally significant relation (P = .048, odds ratio = 0.63, 95% CI = 0.4-1.0). Regarding GA genotype of 15-PGDH rd8752 polymorphism, there was a significant difference between two groups with a 4.5-fold increase in SLE development (P = .0001). The frequency of the A allele was higher in SLE patients than that in controls, showing a 1.4-fold increase in SLE development (P = .018). Conclusion All results showed the protective effects of the dominant model of COX-2 rs2745557 polymorphism and risk factor of 15-PGDH rs8752 polymorphism on SLE development.

OBJECTIVE The aim: Investigate the effect of Lys198Asn polymorphism of the EDN1 gene on ischemic atherothrombotic stroke characteristics. PATIENTS AND METHODS Materials and methods: Venous blood of 170 patients with ischemic atherothrombotic stroke (IAS) and 124 patients without cerebrovascular pathology, who made up the control group, used for the study. Lys198Asn (rs5370) polymorphism of the EDN1 gene was determined by the polymerase chain reaction method followed by restriction fragment length analysis. Statistical analysis was performed using SPSS-17.0. The values of Р < 0.05 were considered reliable. RESULTS Results: An association between the Lys198Asn polymorphism of the EDN1 gene and the IAS development was detected. For Asn/Asn genotype carriers, the risk of IAS developing is 4 times higher than that of homozygotes for the major allele. The association of this polymorphism with the arterial pool, whose atherothrombotic changes lead to the development of IAS, was found in individuals with BMI < 25 kg/m2. Lys198Asn polymorphism also affects the severity of IAS in persons with hypertension and non-smokers. CONCLUSION Conclusion: The Lys198Asn polymorphism of the EDN1 gene influences some characteristics of ischemic stroke.

Restriction fragment length polymorphism (RFLP) is a technology for the molecular characterization of DNA and widely used genome mapping, medical genetics, molecular microbiology and forensics etc. Terminal restriction fragment length polymorphism (T-RFLP), a variant of RFLP is extensively used in environmental microbiology for the microbial community profiling based on the restriction digestion profile of marker gene (16S rRNA, FTHFS etc.) amplicons. At present, there is a lack of a tool which can perform in-silico restriction digestion of a large number of sequences at a time, in an interactive way and as an output produce sequences of the restriction fragments and visualization plot. I have developed a graphical user interface based software “REDigest” for the in-silico restriction digestion analysis for gene or genome sequences. The REDigest software program with a graphical user interface is freely available at https://github.com/abhijeetsingh1704/REDigest.

The 3D organization of chromatin within the nucleus enables dynamic regulation and cell type-specific transcription of the genome. This is true at multiple levels of resolution: on a large scale, with chromosomes occupying distinct volumes (chromosome territories); at the level of individual chromatin fibers, which are organized into compartmentalized domains (e.g., Topologically Associating Domains-TADs), and at the level of short-range chromatin interactions between functional elements of the genome (e.g., enhancer-promoter loops).The widespread availability of Chromosome Conformation Capture (3C)-based high-throughput techniques has been instrumental in advancing our knowledge of chromatin nuclear organization. In particular, Hi-C has the potential to achieve the most comprehensive characterization of chromatin 3D interactions, as it is theoretically able to detect any pair of restriction fragments connected as a result of ligation by proximity.This chapter will illustrate how to compare the chromatin interactome in different experimental conditions, starting from pre-computed Hi-C contact matrices, how to visualize the results, and how to correlate the observed variations in chromatin interaction strength with changes in gene expression.

Background Polycyclic aromatic hydrocarbons (PAHs) exposure had been reported to be a risk factor of mtDNAcn in our early study. However, the effect of metabolic enzymes' genetic polymorphisms on mtDNAcn in PAHs‐Exposure workers has not been fully evaluated. Aim The aim of the study was to explore the effect of metabolic enzymes' genetic polymorphisms on mtDNAcn in PAHs‐Exposure. Methods and Results We investigated the effects of metabolic enzymes' genetic polymorphisms on mtDNAcn among 544 coke oven workers and 238 office staffs. The mtDNAcn of peripheral blood leukocytes was measured using the Real‐time quantitative polymerase chain reaction (PCR) method. PCR and restriction fragment length was used to detect five polymorphisms in GSTT1, GSTM1, GSTP1 rs1695, CYP2E1 rs6413432, and CYP2E1 rs3813867. The mtDNAcn in peripheral blood leukocytes was significantly lower in the exposure group than that in the control group (p

In this protocol, restriction fragments that have been transferred to a membrane by Southern blotting are hybridized to a labeled probe. Methods for stripping the probe from the membrane are also included.

We tested the hypothesis that differences in DNA double-strand break (DSB) repair fidelity underlies differences in individual radiosensitivity and, consequently, normal tissue reactions to radiotherapy. Fibroblast cultures derived from a radio-sensitive (RS) breast cancer patient with grade 3 adverse reactions to radiotherapy were compared with normal control (NC) and hyper-radiosensitive ataxia-telangiectasia mutated (ATM) cells. DSB repair and repair fidelity were studied by Southern blotting and hybridization to Alu repetitive sequence and to a specific 3.2-Mbp NotI restriction fragment on chromosome 21, respectively. Results for DNA repair kinetics using the NotI fidelity assay showed significant differences (P < 0.001) with higher levels of misrepaired (misrejoined and unrejoined) DSBs in RS and ATM compared with NC. At 24-h postradiation, the relative fractions of misrepaired DSBs were 10.64, 23.08, and 44.70% for NC, RS, and ATM, respectively. The Alu assay showed significant (P < 0.05) differences in unrepaired DSBs only between the ATM and both NC and RS at the time points of 12 and 24 h. At 24 h, the relative percentages of DSBs unrepaired were 1.33, 3.43, and 12.13% for NC, RS, and ATM, respectively. The comparison between the two assays indicated an average of 5-fold higher fractions of misrepaired (NotI assay) than unrepaired (Alu assay) DSBs. In conclusion, this patient with increased radiotoxicity displayed more prominent misrepaired than unrepaired DSBs, suggesting that DNA repair fidelity is a potential marker for the adverse reactions to radiotherapy. More studies are required to confirm these results and further develop DSB repair fidelity as a hallmark biomarker for interindividual differences in radiosensitivity.

Piscirickettsia salmonis is a bacterial pathogen that severely impact the aquaculture in several countries as Canada, Scotland, Ireland, Norway, and Chile. It provokes Piscirickettsiosis outbreaks in the marine phase of salmonid farming, resulting in economic losses. The monophyletic genogroup LF-89 and a divergent genogroup EM-90 are responsible for the most severe Piscirickettsiosis outbreaks in Chile. Therefore, the development of methods for quick genotyping of P. salmonis genogroups in field samples is vital for veterinary diagnoses and understanding the population structure of this pathogen. The present study reports the development of a multiplex PCR for genotyping LF-89 and EM-90 genogroups based on comparative genomics of 73 fully sequenced P. salmonis genomes. The results revealed 2,322 sequences shared between 35 LF-89 genomes, 2,280 sequences in the core-genome of 38 EM-90 genomes, and 331 and 534 accessory coding sequences each genogroup, respectively. A total of 1,801 clusters of coding sequences were shared among all tested genomes of P. salmonis (LF-89 and EM-90), with 253 and 291 unique sequences for LF-89 and EM-90 genogroups, respectively. The Multiplex-1 prototype was chosen for reliable genotyping because of differences in annealing temperatures and respective reaction efficiencies. This method also identified the pathogen in field samples infected with LF-89 or EM-90 strains, which is not possible with other methods currently available. Finally, the genome-based multiplex PCR protocol presented in this study is a rapid and affordable alternative to classical sequencing of PCR products and analyzing the length of restriction fragment polymorphisms.

The study analyzed three SNP polymorphisms located in exon 2 of the corticotropin-coding genes (rs110167397, rs381116984, rs109244394), later referred to as CRH1, CRH2, CRH3, respectively. The research was conducted in a Salers cattle herd. Identification of genotypes of individual animals was carried out using polymerase chain reaction - restriction fragments length polymorphism (PCR-RFLP). The heterozygous genotypes of all analyzed polymorphisms they determined the most extensive body mass of the analyzed animals. Other traits, such as cow size, muscularity, hip height and chest girth, were the most favorable for homozygous GG individuals concerning CRH1 polymorphism. Studying the relationship between CRH2 polymorphism and the animals with the genotype TT was preferred for traits such as muscularity and chest girth. It was shown that the CRH3 polymorphism significantly affected the muscularity and higher values of the zoometric measurement (hip height, chest girth) in the case of cows with the genotype CC. Significant differences were found in the study (p ≤ 0.01; p ≤ 0.05).

There are more and more studies on telomere length (TL) and mitochondrial DNA (mtDNA), and it has been proven that these factors play a significant role in the aging of the immune system thereby it is important to understand how it varies in different cell types for more accurate conclusions. The aim of this study was to look into dynamics of mtDNA amount in conjunction with TL in peripheral blood mononuclear cells (PBMC) during aging in comparison with whole blood (WB) cells. Overall, 53 samples were divided into three age groups: 20-39 year age group, 40-59 year age group and 60-79 year age group. MtDNA amount was determined by qPCR TaqMan, and TL was measured by Southern blotting of terminal restriction fragments (TRFs). PBMC had much higher mtDNA copy number (CN) amount than WB samples. Furthermore, with age, it increased in PBMC, while in WB mtDNA CN count did not change. TL in the elderly group was shorter in PBMC fraction than in WB cells. It also looked like that in PBMC TL shortened faster than in WB. In conclusions, it appears that during the aging process both mtDNA CN and TL were more stable in WB than in PBMC fraction where changes were more drastically pronounced, but more studies using larger sample cohorts should be performed to confirm this observation.

Two binucleate Rhizoctonia (BNR) isolates (HLJ-21 and HLJ-57) were recovered from diseased sugar beet seedlings in Heilongjiang Province, Northeastern China in May, 2015. The two isolates were identified as members of the anastomosis group (AG)-W based on morphological characteristics, hyphal anastomosis, polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP), and phylogenetic analysis of the internal transcribed spacer of the ribosomal DNA (rDNA ITS). The optimum temperature for hyphal growth was 25 °C and the mycelia retained a whitish appearance on potato dextrose agar as cultures aged. No sclerotia, however, were observed during the period of culture. Both of the isolates exhibited anastomosis with AG-W, but failed with other reference strains of BNRs. Restriction fragments resulting from the eight enzymes indicated that HLJ-21 and HLJ-57 isolates had the same restriction pattern as AG-W isolates. Phylogenetic analysis also indicated that the rDNA ITS region of the HLJ-21 and HLJ-57 isolates clustered with other AG-W isolates in the same clade. The AG-W isolates from sugar beet and potato were the causal agent of damping-off disease on sugar beet seedlings as well as root disease on wheat and corn seedlings. The AG-W isolates from sugar beet could also induce stem canker disease on potato plants. Notably, the isolates from sugar beet seedlings had a higher virulence on all of the tested crops than those isolates obtained from potato. This appears to be the first report of a AG-W BNR causing sugar beet seedling damping-off disease in China, and perhaps globally.

Alicyclobacillus acidoterrestris is considered to be one of the most important target microorganisms in quality control of heat-processed acidic foods. This species can cause spoilage by forming spores with very high heat resistance; however, no gas production or visible changes occur in the contaminated product during storage. Thus, the differentiation of A. acidoterrestris from other Alicyclobacillus species is of great importance. Τhe present study aims to find a rapid method for the identification of A. acidoterrestris. To achieve this, 78 Alicyclobacillus isolates were subjected to PCR-RFLP (restriction fragment length polymorphism). Specifically, PCR products of amplified V1-V3 region of the 16S rRNA gene were digested with restriction endonuclease HhaI. According to the obtained results, all A. acidoterrestris isolates showed similar restriction fragments of the 16S rRNA gene and different from A. acidocaldarius and A. hesperidum. In conclusion, a single enzyme PCR-RFLP assay was developed and showed rapid, inexpensive and direct identification of Alicyclobacillus isolates. The application of this method will be useful to identify this contaminant in fruit juices.

Alcaligenes javaensis is a food-borne, Gram-negative bacterium, which has the closest relationship with Alcaligenes faecalis commonly causing contamination of clinical equipment and infection on humans. Hence, the development of a biomarker for members of genus Alcaligenes including A.javaensis is needed for early detection of this bacterium. This research aimed to screen the performance of 5 restriction enzymes consisting of EcoRV, Hind III, NotI, SalI, and BgIII as singles and in combinations to produce specific pattern of restricted, genomic DNA fragments of A.javaensis. Total DNA was first extracted from subcultured A.javaensis cells using DNA isolation kit and the obtained DNA isolate was then subjected to enzyme restrictions in vitro. Later, the restricted DNA fragments were evaluated using agarose gel electrophoresis method. Results showed that only the EcoRV-BgIII-HindIII combination within a restriction process of 18 h could produce smaller-sized DNA bands, while those from other combinations (5 enzymes as singles and their 2-combinations) could not do so. As conclucion, the combination of EcoRV-BgIII-HindIII is the most potential among the evaluated endonucleases to be used as a genomic biomarker for A.javaensis. Article DOI: https://dx.doi.org/10.20319/lijhls.2019.51.6272 This work is licensed under the Creative Commons Attribution-Non-commercial 4.0 International License. To view a copy of this license, visit http://creativecommons.org/licenses/by-nc/4.0/ or send a letter to Creative Commons, PO Box 1866, Mountain View, CA 94042, USA.

Background: Alloimmunization is a major problem in transfusion practice due to the clinical complications of the patients and the difficulty of choosing a unit of compatible blood product. Serological methods are widely used in blood banks, but they not always determine the phenotype. Thus, genotyping is an important complement to the serology tool as it allows one to predict the phenotype from deoxyribonucleic acid (DNA) with high accuracy. Objective: To compare the centrifugation gel, microarray, Restriction Fragment Length Polymorphismone PCR (PCR-RFLP) and Sequence-Specific Primer PCR (PCR-SSP) techniques, in terms of cost, reaction time and reliability of the results. Methods: The RHCE, Kidd, Kell and Duffy blood group systems were chosen to determine the approximate cost of each technique, considering the reagents used in both methods and considering only one sample. The time required for the development of each reaction was obtained at the Maringa Regional Blood Center and Immunogenetics Laboratory at the State University of Maringa. Data from Microarray reactions were obtained at the Campinas Blood Center. The results of phenotyping and genotyping of the 16 samples were compiled in a spreadsheet and compared. Results: The PCR-SSP was more economical compared to other methods, and the serological method was faster than the molecular methods. However, all methods proved to be effective and safe in the detection of erythrocyte antigens. Conclusion: Analyzing the advantages and limitations of the molecular and serological methods tested in this study, we note that both are important and complementary. However, the choice of a methodology depends on the reality and needs of each health service. Keywords: Cost analysis, Molecular biology, Serology, Blood group antigens

Introduction Glucose-6-phosphate dehydrogenase (G6PD) deficiency is the most common X-linked enzymopathy, highly prevalent in the areas where malaria is or has been endemic. Prevalence of G6PD deficiency and characterization of G6PD variants in females from previously malaria-endemic areas of northeastern Thailand remain unstudied. Methods Prevalence of G6PD deficiency was determined by a fluorescent spot test (FST), quantitative G6PD activity assay, and multiplex allele-specific (AS)- and restriction fragment length polymorphic (RFLP)-PCR developed for detection of common G6PD variants in the Thai population. Results Prevalence of G6PD deficiency in female samples (n = 355) was 18% by FST, 29.6% by quantitation of G6PD activity, and 28.1% by PCR-based genotyping. The most common variant was G6PD Viangchan (54%), followed by G6PD Canton (11%) and G6PD Union (11%); in addition, a novel heterozygous variant, G6PD Khon Kaen (c.305T>C, p.F102S), was identified. The majority of heterozygotes expressed G6PD activity within the intermediate deficiency range (30-70% median of normal enzyme activity). Conclusion High prevalence of G6PD deficiency was present in females from northeastern Thailand, the majority being due to heterozygosity of G6PD variants. The findings will have a bearing on the inclusion of primaquine in antimalarial-based policies for malaria elimination in populations with a high prevalence of G6PD deficiency.

Thesis Abstract Several cytokines are known to be involved in the pathogenesis of non-Hodgkin Lymphomas (NHL). Given the importance of understanding the genetic predisposition involved in the pathogenesis of NHL, this work was designed to study the impact of IL-10 (-1082 G/A; rs1800896 and -819 C/T; rs1800871), IL-6 (-174 G/C; rs1800795) and CD38 (184C/G; rs6449182) gene polymorphisms on the susceptibility of Egyptians to NHL. Both loci of IL10 were genotyped using polymerase chain reaction- sequence-specific primers (PCR-SSP), IL6 rs1800795 G/C was genotyped using mutagenically separated PCR (MS-PCR) and restriction fragment length polymorphism-PCR (RFLP-PCR) was used to genotype CD38 (rs6449182 C/G) in 100 Egyptian patients and 119 controls. Plasma levels of IL-6 and IL-10 were measured using enzyme-linked immunosorbent assay (ELISA). An insignificant change in IL-10 (rs1800896 and rs1800871) genotypes were recorded. GT haplotype was significantly elevated.

Diagnosis of trichinellosis and the identification of Trichinella spp. have taken many forms over the years. Given the absence of morphological characters to advance that goal, science turned to immunological, biochemical, and molecular techniques. Immunological methods have worked well for identifying Trichinella-infected hosts; however, in general, methods have not been developed for identifying individual species and genotypes other than for differentiating among the encapsulated and nonencapsulated clades. This has been hindered by the strong immunodominant tyvelose antigens that are conserved among all members of the genus. Biochemical markers, predominantly isoenzyme patterns, have been more successful at defining genotypes; however, the assays tend to be labor intensive, work best on sizeable samples, and generally require large control datasets for comparative purposes. Thus over time, the industry has turned to molecular techniques, with the earliest method, restriction fragment polymorphism, surfacing in the 1980s. Over time, species identification has followed the evolution of molecular techniques from macroscales to the identification of individual worms via next-generation sequencing. Herein we review the advancement of molecular approaches to studying Trichinella spp. not only for diagnosing and differentiating among genotypes, but also as a prelude to expanding our understanding of population genetics and the evolution of the genus.

The morphological identification of mites entails great challenges. Characteristics such as dorsal setae and aedeagus are widely used, but they show variations between populations, and the technique is time consuming and demands specialized taxonomic expertise that is difficult to access. A successful alternative has been to exploit a region of the mitochondrial cytochrome oxidase I (COI) gene to classify specimens to the species level. We analyzed the COI sequences of four mite species associated with cassava and classified them definitively by detailed morphological examinations. We then developed an identification kit based on the restriction fragment length polymorphism–polymerase chain reaction of subunit I of the COI gene focused on the three restriction enzymes AseI, MboII, and ApoI. This set of enzymes permitted the simple, accurate identification of Mononychellus caribbeanae, M. tanajoa, M. mcgregori, and Tetranychus urticae, rapidly and with few resources. This kit could be a vital tool for the surveillance and monitoring of mite pests in cassava crop protection programs in Africa, Asia, and Latin America.

Genome-wide chromosome conformation capture (3C)-based high-throughput sequencing (Hi-C) has enabled identification of genome-wide chromatin loops. Because the Hi-C map with restriction fragment resolution is intrinsically associated with sparsity and stochastic noise, Hi-C data are usually binned at particular intervals; however, the binning method has limited reliability, especially at high resolution. Here, we describe a new method called HiCORE, which provides simple pipelines and algorithms to overcome the limitations of single-layered binning and predict core chromatin regions with 3D physical interactions. In this approach, multiple layers of binning with slightly shifted genome coverage are generated, and interacting bins at each layer are integrated to infer narrower regions of chromatin interactions. HiCORE predicts chromatin looping regions with higher resolution and contributes to the identification of the precise positions of potential genomic elements.Author SummaryThe Hi-C analysis has enabled to obtain information on 3D interaction of genomes. While various approaches have been developed for the identification of reliable chromatin loops, binning methods have been limitedly improved. We here developed HiCORE algorithm that generates multiple layers of bin-array and specifies core chromatin regions with 3D interactions. We validated our algorithm and provided advantages over conventional binning method. Overall, HiCORE facilitates to predict chromatin loops with higher resolution and reliability, which is particularly relevant in analysis of small genomes.

Background This study describes the biodiversity and properties of Bovine leukemia virus in Western Siberia. This paper explores the effect of different genotypes of the env gene of the cattle leukemia virus on hematological parameters of infected animals. The researchers focused on exploring the polymorphism of the env gene and, in doing so, discovered the new genotypes Ia and Ib, which differ from genotype I. Several hypotheses on the origin of the different genotypes in Siberia are discussed. Results We obtained varying length of the restriction fragments for genotypes I. Additionally using restrictase Hae III were received fragments was named genotype Ia, and genotype Ib. There are 2.57 ± 0.55% (20 out of 779) samples of genotype Ib which does not differ significantly from 1% (χ2 = 2.46). Other genotypes were observed in the cattle of Siberia as wild type genotypes (their frequency varied from 17.84 to 32.73%). The maximum viral load was observed in animals with the II and IV viral genotypes (1000–1400 viral particles per 1000 healthy cells), and the minimum viral load was observed animals with genotype Ib (from 700 to 900 viral particles per 1000 healthy cells). Conclusions The probability of the direct introduction of genotype II from South America to Siberia is extremely small and it is more likely that the strain originated independently in an autonomous population with its distribution also occurring independently. A new variety of genotype I (Ib) was found, which can be both a neoplasm and a relict strain.

Our 25 years of experience in carrier diagnosis of hemophilia A (HA) and B (HB) in Mexican population comprises linkage analysis of intragenic F8/F9 neutral variants along with, in severe HA (SHA), detection of F8 int22h and int1h inversions. In symptomatic carriers (SCs) we explored Lyonization to explain their symtomatology. From a DNA-Bank of 3,000 samples, intragenic restriction fragment length (RFLPs) and short tandem repeats (STRs) of F8/F9 genes were assessed by PCR-PAGE and GeneScan. In SHA patients, F8 inversions were detected by inverse shifting-PCR/diagnostic and complementary tests. In SCs, we evaluated hemorrhagic symptoms, clotting FVIII/FIX and X-chromosome inactivation (XCI) patterns were assessed by HUMARA assay and the search of XIST promoter pathogenic variants. Informativeness of linkage analysis for HA carrier diagnosis with RFLP's/STR's increased to 74% and reached 80% with five RFLPs for HB. Combined Inv22/Inv1 diagnosed 113 possible carriers, three de novo Inv22-1, and confirmed 45 mothers as obligate or sporadic carriers. Among 21 SCs, four showed extreme skewed XCI pattern (~80:20) but had normal karyotype and no C43G pathogenic variant in XIST promoter. Clotting FVIII/FIX correlated with the active X in leukocytes. Our data integrate the largest comprehensive research worldwide on the molecular diagnosis of HA and HB carriers in terms of the number of studied and diagnosed cases, in addition to the genetic analysis in SCs. Intragenic RFLPs and STRs of F8/F9 genes along with F8 int22h/int1h inversions in SHA emerge as optimal variants for molecular diagnosis in Mexican population. In counseling SCs, inheritance of skewed X-inactivation should be considered.

The BioBrick standard makes possible iterated pairwise assembly of cloned parts without any depletion of unique restriction sites. Every part that conforms to the standard is compatible with every other part, thereby fostering a worldwide user community. The assembly methods, however, are labor intensive or inefficient compared to some newer ones so the standard may be falling out of favor. An easier way to assemble BioBricks is described herein. Plasmids encoding BioBrick parts are purified from Escherichia coli cells that express a foreign site-specific DNA methyltransferase, so that each is subsequently protected in vitro from the activity of a particular restriction endonuclease. Each plasmid is double-digested and all resulting restriction fragments are ligated together without gel purification. The ligation products are subsequently double-digested with another pair of restriction endonucleases so only the desired insert-recipient vector construct retains the capacity to transform E. coli. This 4R/2M BioBrick assembly protocol is more efficient and accurate than established workflows including 3A assembly. It is also much easier than gel purification to miniaturize, automate and perform more assembly reactions in parallel. As such, it should streamline DNA assembly for the existing community of BioBrick users, and possibly encourage others to join.

The identification of the type I junction allowed us to establish the correct permutation of the circularly permuted type I map vis a vis the parental dihydrofolate reductase (DHFR) locus. The high copy number made it possible to visualize restriction fragments arising from the amplicons in digests separated on ethidium bromide-stained agarose gels. The earliest detectable DHFR amplicons were almost invariably located on chromosome arm as one of the parental, single copy loci. Initial recombinant clones from the amplicons in CHOC 400 cells were isolated from a cosmid library prepared in the vector pHC79. In quantitative hybridization studies, the type I sequence was shown to represent only 5–10% of the amplicons in CHOC 400 cells. In situ hybridization studies on the relatively early stages of DHFR gene amplification in Chinese hamster ovary cells also suggest that initial amplicons can be extremely large.

The chromatin organization in the 3D nuclear space is essential for genome functionality. This spatial organization encompasses different topologies at diverse scale lengths with chromosomes occupying distinct volumes and individual chromosomes folding into compartments, inside which the chromatin fiber is packed in large domains (as the topologically associating domains, TADs) and forms short-range interactions (as enhancer-promoter loops). The widespread adoption of high-throughput techniques derived from chromosome conformation capture (3C) has been instrumental in investigating the nuclear organization of chromatin. In particular, Hi-C has the potential to achieve the most comprehensive characterization of chromatin 3D structures, as in principle it can detect any pair of restriction fragments connected as a result of ligation by proximity. However, the analysis of the enormous amount of genomic data produced by Hi-C techniques requires the application of complex, multistep computational procedures that may constitute a difficult task also for expert computational biologists. In this chapter, we describe the computational analysis of Hi-C data obtained from the lymphoblastoid cell line GM12878, detailing the processing of raw data, the generation and normalization of the Hi-C contact map, the detection of TADs and chromatin interactions, and their visualization and annotation.

Background Telomeres, nucleoprotein structures comprising short tandem repeats and delimiting the ends of linear eukaryotic chromosomes, play an important role in the maintenance of genome stability. Therefore, the determination of the length of telomeres is of high importance for many studies. Over the last years, new methods for the analysis of the length of telomeres have been developed, including those based on PCR or analysis of NGS data. Despite that, terminal restriction fragment (TRF) method remains the gold standard to this day. However, this method lacks universally accepted and precise tool capable to analyse and statistically evaluate TRF results. Results To standardize the processing of TRF results, we have developed WALTER, an online toolset allowing rapid, reproducible, and user-friendly analysis including statistical evaluation of the data. Given its web-based nature, it provides an easily accessible way to analyse TRF data without any need to install additional software. Conclusions WALTER represents a major upgrade from currently available tools for the image processing of TRF scans. This toolset enables a rapid, highly reproducible, and user-friendly evaluation of almost any TRF scan including in-house statistical evaluation of the data. WALTER platform together with user manual describing the evaluation of TRF scans in detail and presenting tips and troubleshooting, as well as test data to demo the software are available at https://www.ceitec.eu/chromatin-molecular-complexes-jiri-fajkus/rg51/tab?tabId=125#WALTER and the source code at https://github.com/mlyc93/WALTER.

Background Acute myeloid leukemia (AML) is a malignant hematological neoplasm of myeloid progenitor cells. Mutations of FLT3 in its tyrosine kinase domain (FLT3-TKD) are found in ~ 8% of patients with AML, with D835Y as the most common substitution. This mutation activates survival signals that drives the disease and is resistant to the first generation FLT3 inhibitors. Development of a highly sensitive method to detect FLT3D835Y is important to direct therapeutic options, predict prognosis, and monitor minimal residual disease in patients with AML. Methods and results In the present study, we developed a highly sensitive FLT3D835Y detection method by using the restriction fragment nested allele-specific PCR technique. The method consists of three steps: 1) initial amplification of DNA samples with PCR primers surrounding the FLT3D835Y mutation site, 2) digestion of the PCR products with restriction enzyme EcoRV that only cleaves the wild type allele, and 3) detection of FLT3D835Y by allele-specific PCR with nested primers. We were able to detect FLT3D835Y with a sensitivity of 0.001% by using purified plasmid DNAs and blood cell DNAs containing known proportions of FLT3D835Y. We analyzed blood cell DNA samples from 64 patients with AML and found six FLT3D835Y-positive cases, two of which could not be detected by conventional DNA sequencing methods. Importantly, the method was able to detect FLT3D835Y in a sample collected from a relapsed patient while the patient was in complete remission with negative MRD determined by flow cytometry. Therefore, our RFN-AS-PCR detected MRD after treatment that was missed by flow cytometry and Sanger DNA sequencing, by conventional methods. Conclusions We have developed a simple and highly sensitive method that will allow for detection of FLT3D835Y at a very low level. This method may have major clinical implications for treatment of AML.

Ginseng Radix, the dried root of Panax ginseng, is easily confused with other herbs of the Panax species by appearance. In Chinese medicine preparations, it is difficult to identify misuse of Ginseng Radix. In this study, the nested PCR and DNA sequencing methods were established for the identification of Ginseng Radix, whereas the combination of nested PCR and restriction fragment length polymorphism (RFLP) could differentiate Ginseng Radix from other Panax species and thus certify the use of impure raw material of Ginseng Radix in Chinese medicine preparations. Nested PCR and DNA sequencing methods were applied to verify ginseng ingredients in the preparations. Of the 58 samples analyzed, 48 samples contained P. ginseng, 8 P. quinguefolius, and 2 P. notoginseng. Utilizing restriction enzyme BstYI and DdeI, nested PCR products of the samples were digested for restriction fragment lengths of polymorphism (RFLP). It was proven that 23 of the 58 samples contained a mixture of Ginseng Radix and Panacis quinquefolii Radix. Twenty-five samples contained pure raw material of Ginseng Radix. Ginseng components of 10 samples were confirmed using a substitute of P. quinguefolius or P. notoginseng. The RFLP results were consistent with those of the sequencing method results.

Sequence analysis and polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) technique were used to identify the species of Thunnus thynnus, T. alalunga, T. obesus and T. albacares. Genetic variation of 376bp fragments in mitochondrial cytochrome b gene of 4 Thunnus species captured from northern Pacific Ocean, northern Atlantic Ocean and Indian Ocean was analyzed. The restriction enzymes and restriction fragment length pattern in each Thunnus species were obtained. It was found that the 376bp fragment of cytochrome b gene in T. obesus showed the obvious divergence of 4.235% different from other 3 species. The highest interspecific similarity ranged 98.687~99.674% between T. thynnus and T. alalunga. PCR-RFLP by using 3 specific restriction enzymes Bsp1286, Hinc Ⅱ and Rsa Ⅰ could precisely and quickly identify the species of the 4 common Thunnus species. Furthermore, this technique successfully detected the species of 12 samples of commercial tuna fillets (sashimi).

Astragali Radix are originated from Astragalus membranaceus and A. membranaceus var. mongholicus. However, Hedysarum Radix, the dried root of Hedysarum polybotrys, is often used as a substitute in Taiwan. In Chinese medicinal preparations, authenticity of Astragali Radix is difficult to be certified. The nested PCR and DNA sequencing method was used to identify Astragali Radix and Hedysarum Radix in preparation samples. Primer sets were designed for detecting Astragali Radix and Hedysarum Radix. The total DNA of all samples was extracted. Internal transcribed spacer (ITS) regions of the DNA samples were amplified by nested PCR and the sequences received from an auto-sequencer. We compared ITS sequences of the preparation samples with the standard to check whether Astragali Radix or Hedysarum Radix was in the preparation samples. Results indicated that 20 out of 30 samples contained a mixture of Astragali Radix and Hedysarum Radix, five Hedysarum Radix as substitute and five Astragali Radix. The combination of nested PCR and restriction fragment lengths polymorphism (RFLP) was employed to rapidly differentiate Astragalus membranaceus, A. membranaceus var. mongholicus and hybrid. The nested PCR and DNA sequencing method successfully identified Astragali Radix and Hedysarum Radix in Chinese medicine preparations.

Quantitative PCR is frequently used to measure average telomere length (TL) relative to the TL of a reference DNA sample of the investigator’s choosing. This makes comparisons of TLs across studies and laboratories difficult. Here we demonstrate that a single synthetic single-stranded dual-template oligonucleotide (DTO) containing both a telomere repeat sequence (T) and a segment of the human beta-globin (HBB) single copy gene (S) can be used as a universal reference standard for monochrome multiplex quantitative PCR (MMqPCR) measurements of average TL using SYBR Green I as the only fluorescent reporter dye. A set of twelve concentrations of the DTO is prepared by serial 3-fold dilutions, to a lowest concentration of ~20 copies per μl. The 5 highest concentrations are used for the T standard curve, and the 5 lowest concentrations are used for the S standard curve. For each reaction 5 μl containing approximately 3 ng of genomic DNA (or one of the DTO dilutions) is mixed with 5 μl of a 2x MasterMix containing the primers for T and S amplification, and MMqPCR is performed. The design of the primers and thermal cycling profile allows all T amplification signals to be collected before exponential amplification of the S signal begins. Exponential amplification from S is then carried out in a temperature range that keeps the telomere product fully melted and therefore unable to influence the S amplification signal. The T value for each DNA sample is the Standard Curve DTO dilution that contains the same number of copies of the telomere sequence as the experimental sample, and the S value is the DTO dilution that contains the same number of copies of the single copy gene sequence as the experimental sample. Dividing the first dilution by the second dilution yields an absolute T/S ratio, since it is expressed relative to the fixed 1:1 T/S ratio that is built into the DTO by design. Absolute T/S ratios for average TL in 48 human DNA samples determined by this method correlated strongly with mean Terminal Restriction Fragment (mTRF) lengths for the same DNA samples determined by the Southern Blot method (R-squared = 0.801). This DTO and the accompanying protocol may facilitate the standardization of average telomere length measurements and analyses across laboratories.

An investigation of sex-specific loci may provide important insights into fish sex determination strategies. This may be useful for biotechnological purposes, for example, to produce all-male or all-female fish for commercial breeding. The North African catfish species, Clarias gariepinus, has been widely adopted for aquaculture because its superior growth and disease resistance render the species suitable for hybridization with other catfish to improve the productivity and quality of fish meat. This species has either a ZZ/ZW or XX/XY sex determination system. Here, we investigate and characterize these systems using high-throughput genome complexity reduction sequencing as Diversity Arrays Technology. This approach was effective in identifying moderately sex-linked loci with both single-nucleotide polymorphisms (SNPs) and restriction fragment presence/absence (PA) markers in 30 perfectly sexed individuals of C. gariepinus. However, SNPs based markers were not found in this study. In total, 41 loci met the criteria for being moderately male-linked (with male vs. female ratios 80:20 and 70:30), while 25 loci were found to be moderately linked to female sex. No strictly male- or female-linked loci were detected. Seven moderately male-linked loci were partially homologous to some classes of transposable elements and three moderately male-linked loci were partially homologous to functional genes. Our data showed that the male heterogametic XX/XY sex determination system should co-exist with the ZZ/ZW system in C. gariepinus. Our finding of the co-existence of XX/XY and ZZ/ZW systems can be applied to benefit commercial breeding of this species in Thailand. This approach using moderately sex-linked loci provides a solid baseline for revealing sex determination mechanisms and identify potential sex determination regions in catfish, allowing further investigation of genetic improvements in breeding programs.

Telomere length is measured using Southern blotting of the chromosomal terminal restriction fragments (TRFs) released by endonuclease digestion in cells from yeast to human. In the budding yeast Saccharomyces cerevisiae, XhoI or PstI is applied to cut the subtelomere Y' element and release TRFs from the 17 subtelomeres. However, telomeres from other 15 X-element-only subtelomeres are omitted from analysis. Here, we report a method for measuring all 32 telomeres in S. cerevisiae using the endonuclease MmeI. Based on analyses of the endonuclease cleavage sites, we found that the TRFs generated by MmeI displayed two distinguishable bands in the sizes of ~500 and ~700 bp comprising telomeres (300 bp) and subtelomeres (200-400 bp). The modified MmeI-restricted TRF (mTRF) method recapitulated telomere shortening and lengthening caused by deficiencies of YKu and Rif1 respectively in S. cerevisiae. Furthermore, we found that mTRF was also applicable to telomere length analysis in S. paradoxus strains. These results demonstrate a useful tool for simultaneous detection of telomeres from all chromosomal ends with both X-element-only and Y'-element subtelomeres in S. cerevisiae species.

The use of analytical methodologies based on DNA (e.g. PCR-RFLP) to determine the authenticity of different types of meat products after the initial labeling is pivotal to avoid fraudulent practices due to the increased rates of meat consumption. Our PCR-RFLP of mitochondrial 12S rRNA gene using restriction enzymes (AluI and ApoI) aimed to confirm the accuracy of the meat species labeling, based on fresh and processed meat collected in central markets along the main cities in the Amazonas Region (Bagua, Bagua Grande, Chachapoyas, Luya, Pedro Ruiz, Rodriguez de Mendoza). Our analyses qualitatively identified and differentiated three commercial species of fresh meat (bovine, porcine, ovine) and also found the substitution of goat by sheep meat. Regarding processed meat, its composition was uncertain and further analyses should be addressed to determine the meat origin. Monitoring using DNA-based analytical methods of meat trade is suggested to determine fraudulent practices, such as species substitution in markets along regions of Peru. Resumo O uso de metodologias analíticas baseadas em DNA (PCR-RFLP) para determinar, após a rotulagem inicial, a autenticidade de diferentes tipos de produtos derivados de carne é essencial para evitar práticas fraudulentas, tendo em vista o crescente aumento do consumo de carne. Nossa análise por PCR-RFLP do gene mitocondrial 12S rRNA usando enzimas de restrição (AluI e ApoI) teve como objetivo confirmar a precisão da rotulagem de espécies de carne animal, com base em amostras de carne fresca e processada, coletadas em mercados centrais nas principais cidades da região do Amazonas (Bagua, Bagua Grande, Chachapoyas, Luya, Pedro Ruiz, Rodriguez de Mendoza). Nossas análises identificaram e diferenciaram qualitativamente três espécies comerciais de carne fresca (bovina, suína, ovina) e também encontraram a substituição de espécies de cabra por carne ovina. Em relação à carne processada, sua composição mostrou-se incerta e análises adicionais devem ser feitas para determinar a origem desta. Sugere-se o monitoramento do comércio de carne usando métodos analíticos baseados no DNA para determinar práticas fraudulentas como substituição de espécies nos mercados ao longo das regiões do Peru.

Background A large number of studies have suggested a correlation between the status of telomeres and disease risk. High-throughput quantitative fluorescence in situ hybridization (HT Q-FISH) is a highly accurate telomere measurement technique that can be applied to the study of large cell populations. Here we describe the analytical performance testing and validation of Telomere Analysis Technology (TAT®), a laboratory-developed HT Q-FISH-based methodology that includes HT imaging and software workflows that provide a highly detailed view of telomere populations. Methods TAT was developed for the analysis of telomeres in peripheral blood mononuclear cells (PBMCs). TAT was compared with Terminal Restriction Fragment (TRF) length analysis, and tested for accuracy, precision, limits of detection (LOD) and specificity, reportable range and reference range. Results Using 6 different lymphocyte cell lines, we found a high correlation between TAT and TRF for telomere length (R2 ≥ 0.99). The standard variation (assay error) of TAT was 454 base pairs, and the limit of detection of 800 base pairs. A standard curve was constructed to cover human median reportable range values and defined its lower limit at 4700 bp and upper limits at 14,400 bp. Using TAT, up to 223 telomere associated variables (TAVs) can be obtained from a single sample. A pilot, population study, of telomere analysis using TAT revealed high accuracy and reliability of the methodology. Conclusions Analytical validation of TAT shows that is a robust and reliable technique for the characterization of a detailed telomere profile in large cell populations. The combination of high-throughput imaging and software workflows allows for the collection of a large number of telomere-associated variables from each sample, which can then be used in epidemiological and clinical studies.

Hi-C (high throughput chromosome conformation capture) is a powerful tool designed to explore the genome-wide collection of chromosomal contacts within a cell population. However, the highly complex nature of Hi-C libraries often precludes the robust identification of interactions between individual restriction fragments with high resolution and statistical significance. This challenge led to the incorporation of sequence capture technology into Hi-C protocols, giving rise to a Capture Hi-C methodology. In this chapter, we review the general principles of the technique and provide a view emanating from our experience employing this approach to study the dynamics of episomal viral DNA contacts during specific stages of the viral life cycle.