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Gel image analyses are often difficult to reproduce, as the most commonly used software, the ImageJ Gels plugin, does not automatically record any steps in the analysis process. This protocol provides detailed steps for image analysis using IOCBIO Gel software with western blot as an example; however, the protocol is applicable to all images obtained by electrophoresis, such as Southern blotting, northern blotting, and isoelectric focusing. IOCBIO Gel allows multiple sample analyses, linking the original image to all the operations performed on it, which can be stored in a central database or on a PC, ensuring ease of access and the possibility to perform corrections at each analysis stage. In addition, IOCBIO Gel is lightweight, with only minimal computer requirements. Key features • Free and open-source software for analyzing gel images. • Reproducibility. • Can be used with images obtained by electrophoresis, such as western blotting, Southern blotting, isoelectric focusing, and more., Competing Interests: Competing interestsThe authors declare that they have no competing interests., (©Copyright : © 2024 The Authors; This is an open access article under the CC BY license.)

RFC1 disease, caused by biallelic repeat expansion in RFC1, is clinically heterogeneous in terms of age of onset, disease progression and phenotype. We investigated the role of the repeat size in influencing clinical variables in RFC1 disease. We also assessed the presence and role of meiotic and somatic instability of the repeat. In this study, we identified 553 patients carrying biallelic RFC1 expansions and measured the repeat expansion size in 392 cases. Pearson's coefficient was calculated to assess the correlation between the repeat size and age at disease onset. A Cox model with robust cluster standard errors was adopted to describe the effect of repeat size on age at disease onset, on age at onset of each individual symptoms, and on disease progression. A quasi-Poisson regression model was used to analyse the relationship between phenotype and repeat size. We performed multivariate linear regression to assess the association of the repeat size with the degree of cerebellar atrophy. Meiotic stability was assessed by Southern blotting on first-degree relatives of 27 probands. Finally, somatic instability was investigated by optical genome mapping on cerebellar and frontal cortex and unaffected peripheral tissue from four post-mortem cases. A larger repeat size of both smaller and larger allele was associated with an earlier age at neurological onset [smaller allele hazard ratio (HR) = 2.06, P < 0.001; larger allele HR = 1.53, P < 0.001] and with a higher hazard of developing disabling symptoms, such as dysarthria or dysphagia (smaller allele HR = 3.40, P < 0.001; larger allele HR = 1.71, P = 0.002) or loss of independent walking (smaller allele HR = 2.78, P < 0.001; larger allele HR = 1.60; P < 0.001) earlier in disease course. Patients with more complex phenotypes carried larger expansions [smaller allele: complex neuropathy rate ratio (RR) = 1.30, P = 0.003; cerebellar ataxia, neuropathy and vestibular areflexia syndrome (CANVAS) RR = 1.34, P < 0.001; larger allele: complex neuropathy RR = 1.33, P = 0.008; CANVAS RR = 1.31, P = 0.009]. Furthermore, larger repeat expansions in the smaller allele were associated with more pronounced cerebellar vermis atrophy (lobules I-V β = -1.06, P < 0.001; lobules VI-VII β = -0.34, P = 0.005). The repeat did not show significant instability during vertical transmission and across different tissues and brain regions. RFC1 repeat size, particularly of the smaller allele, is one of the determinants of variability in RFC1 disease and represents a key prognostic factor to predict disease onset, phenotype and severity. Assessing the repeat size is warranted as part of the diagnostic test for RFC1 expansion., (© The Author(s) 2024. Published by Oxford University Press on behalf of the Guarantors of Brain.)

Telomeres are located at the ends of chromosomes and have specific sequences with a distinctive structure that safeguards genes. They possess capping structures that protect chromosome ends from fusion events and ensure chromosome stability. Telomeres shorten in length during each cycle of cell division. When this length reaches a certain threshold, it can lead to genomic instability, thus being implicated in various diseases, including cancer and neurodegenerative disorders. The possibility of telomeres serving as a biomarker for aging and age-related disease is being explored, and their significance is still under study. This is because post-mitotic cells, which are mature cells that do not undergo mitosis, do not experience telomere shortening due to age. Instead, other causes, for example, exposure to oxidative stress, can directly damage the telomeres, causing genomic instability. Nonetheless, a general agreement has been established that measuring telomere length offers valuable insights and forms a crucial foundation for analyzing gene expression and epigenetic data. Numerous approaches have been developed to accurately measure telomere lengths. In this review, we summarize various methods and their advantages and limitations for assessing telomere length., Competing Interests: The authors have no potential conflicts of interest to disclose. The funder played no role in the study’s design, data collection, analysis, interpretation of the data, writing of the report, or the decision to submit the report for publication., (© 2024 The Authors.)

Meiosis is a process through which diploid cells divide into haploid cells, thus promoting genetic diversity. This diversity arises from the formation of genetic crossovers (COs) that repair DNA double-strand breaks (DSBs), through homologous recombination (HR). Deficiencies in HR can lead to chromosomal abnormality resulting from chromosomal nondisjunction, and genetic disorders. Therefore, investigating the mechanisms underlying effective HR is crucial for reducing genome instability. Budding yeast serves as an ideal model for studying HR mechanisms due to its amenability to gene modifications and the ease of inducing synchronized meiosis to yield four spores. During meiosis, at the DNA level, programmed DSBs are repaired as COs or non-crossovers (NCOs) through structural alterations in the nascent D-loop, involving single-end invasions (SEIs) and double-Holliday junctions (dHJs). This repair occurs using homologous templates rather than sister templates. This protocol, using Southern blotting, allows for the analysis and monitoring of changes in DNA structures in the recombination process. One-dimensional (1D) gel electrophoresis is employed to detect DSBs, COs, and NCOs, while two-dimensional (2D) gel electrophoresis is utilized to identify joint molecules (JMs). Therefore, physical analysis is considered the most effective method for investigating the HR mechanism. Our protocol provides more comprehensive information than previous reports by introducing conditions for obtaining a greater number of cells from synchronized yeast and a method that can analyze not only meiotic/mitotic recombination but also mitotic replication., (© 2023. The Author(s), under exclusive licence to Microbiological Society of Korea.)

Background: Current solutions for the analysis of Western Blot images lack either transparency and reproducibility or can be tedious to use if one has to ensure the reproducibility of the analysis., Results: Here, we present an open-source gel image analysis program, IOCBIO Gel. It is designed to simplify image analysis and link the analysis results with the metadata describing the measurements. The software runs on all major desktop operating systems. It allows one to use it in either a single-researcher environment with local storage of the data or in a multiple-researcher environment using a central database to facilitate data sharing within the research team and beyond. By recording the original image and all operations performed on it, such as image cropping, subtraction of background, sample lane selection, and integration boundaries, the software ensures the reproducibility of the analysis and simplifies making corrections at any stage of the research. The analysis results are available either through direct access to the database used to store it or through the export of the relevant data., Conclusions: The software is not only limited to Western Blot image analysis and can be used to analyze images obtained as a part of many other widely used biochemical techniques such as isoelectric focusing. By recording the original data and all the analysis steps, the program improves reproducibility in the analysis and contributes to the implementation of FAIR principles in the related fields., (© 2023. BioMed Central Ltd., part of Springer Nature.)

A recessive Short Tandem Repeat expansion in RFC1 has been found to be associated with cerebellar ataxia, neuropathy and vestibular areflexia syndrome (CANVAS), and to be a frequent cause of late onset ataxia and sensory neuropathy. The usual procedure for sizing these expansions is based on Southern Blotting (SB), a time-consuming and a relatively imprecise technique. In this paper, we compare SB with Optical Genome Mapping (OGM), a method for detecting Structural Variants (SVs) based on the measurement of distances between fluorescently labelled probes, for the diagnosis of RFC1 CANVAS and disease spectrum. The two methods are applied to 17 CANVAS patients' blood samples and resulting sizes compared, showing a good agreement. Further, long-read sequencing is used for two patients to investigate the agreement of sizes with either SB or OGM. Our study concludes that OGM represents a viable alternative to SB, allowing for a simpler technique, a more precise sizing of the expansion and ability to expand analysis of SV in the entire genome as opposed to SB which is a locus specific method.

Telomere length (TL) limits somatic cell replication. However, the shortest among the telomeres in each nucleus, not mean TL, is thought to induce replicative senescence. Researchers have relied on Southern blotting (SB), and techniques calibrated by SB, for precise measurements of TL in epidemiological studies. However, SB provides little information on the shortest telomeres among the 92 telomeres in the nucleus of human somatic cells. Therefore, little is known about the accumulation of short telomeres with age, or whether it limits the human lifespan. To fill this knowledge void, we used the Telomere-Shortest-Length-Assay (TeSLA), a method that tallies and measures single telomeres of all chromosomes. We charted the age-dependent buildup of short telomeres (<3 kb) in human hematopoietic cells from 334 individuals (birth-89 years) from the general population, and 18 patients with dyskeratosis congenita-telomere biology disorders (DC/TBDs), whose hematopoietic cells have presumably reached or are close to their replicative limit. For comparison, we also measured TL with SB. We found that in hematopoietic cells, the buildup of short telomeres occurs in parallel with the shortening with age of mean TL. However, the proportion of short telomeres was lower in octogenarians from the general population than in patients with DC/TBDs. At any age, mean TL was longer and the proportion of short telomeres lower in females than in males. We conclude that though converging to the TL-mediated replicative limit, hematopoietic cell telomeres are unlikely to reach this limit during the lifespan of most contemporary humans., (© 2023 The Authors. Aging Cell published by Anatomical Society and John Wiley & Sons Ltd.)

Tomato yellow leaf curl virus (TYLCV) is a global spreading begomovirus that is exerting a major restraint on global tomato production. In this transgenic approach, an RNA interference (RNAi)-based construct consisting of sequences of an artificial microRNA (amiRNA), a group of small RNA molecules necessary for plant cell development, signal transduction, and stimulus to biotic and abiotic disease was engineered targeting the AC1/Rep gene of the Oman strain of TYLCV-OM. The Rep-amiRNA constructs presented an effective approach in regulating the expression of the Rep gene against TYLCV as a silencing target to create transgenic Solanum lycopersicum L. plant tolerance against TYLCV infection. Molecular diagnosis by PCR followed by a Southern hybridization analysis were performed to confirm the effectiveness of agrobacterium-mediated transformation in T0/T1-transformed plants. A substantial decrease in virus replication was observed when T1 transgenic tomato plants were challenged with the TYLCV-OM infectious construct. Although natural resistance options against TYLCV infection are not accessible, the current study proposes that genetically transformed tomato plants expressing amiRNA could be a potential approach for engineering tolerance in plants against TYLCV infection and conceivably for the inhibition of viral diseases against different strains of whitefly-transmitted begomoviruses in Oman., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Al-Roshdi, Ammara, Khan, Al-Sadi and Shahid.)

Repeat expansion diseases, including fragile X syndrome, Huntington's disease, and C9orf72 -related motor neuron disease and frontotemporal dementia, are a group of disorders associated with polymorphic expansions of tandem repeat nucleotide sequences. These expansions are highly repetitive and often hundreds to thousands of repeats in length, making accurate identification and determination of repeat length via PCR or sequencing challenging. Here we describe a protocol for monitoring repeat length in Drosophila models carrying 1,000 repeat C9orf72 -related dipeptide repeat transgenes using Southern blotting. This protocol has been used regularly to check the length of these lines for over 100 generations with robust and repeatable results and can be implemented for monitoring any repeat expansion in Drosophila ., Competing Interests: Competing interestsThe authors declare no competing interests, (Copyright © 2022 The Authors; exclusive licensee Bio-protocol LLC.)

Trichomoniasis is the most common nonviral sexually transmitted disease; it is caused by Trichomonas vaginalis and seriously threatens human reproductive health. Telomeres are specialised DNA-protein complexes at the ends of chromosomes that have a protective function. The aim of the present study was to identify and characterise the telomeric DNA of T. vaginalis-which has not been previously reported-by multiple molecular methods including sequencing, the Bal nuclease (BAL) 31 nuclease assay, fluorescence in situ hybridisation (FISH), and Southern blotting. We found numerous repeated units of TTTTAGGG in T. vaginalis genomic DNA digested with S1 nuclease in combination with XbaI restriction enzyme. The (TTTTAGGG) n tandem repeats were also highly sensitive to BAL 31 exonuclease digestion. We confirmed that the (TTTTAGGG) n repeats were located at the end of T. vaginalis chromosomes by FISH. Restriction enzyme digestion combined with Southern blotting using a digoxigenin-labelled (TTTTAGGG) 5 probe showed that the T. vaginalis telomeric DNA length varied from 1.0 to 1.5 kb. This is the first report on the telomeric DNA sequence of T. vaginalis which includes the length and distribution on chromosomes; our findings lay a foundation for further study on telomere maintenance mechanisms in T. vaginalis., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Background: Conventional diagnosis of fragile X syndrome (FXS) is based on a combination of fragment analysis (FA) and Southern blotting (SB); however, this diagnostic approach is time- and labor-intensive and has pitfalls such as the possibility of missing large number alleles. Triplet repeat primed PCR (TP-PCR) is a current alternative used to overcome these limitations. We evaluated the diagnostic usefulness of TP-PCR compared with the conventional diagnostic protocol consisting of FA and/or SB in terms of allele categorization, repeat number correlation, and zygosity concordance in female genetic carriers., Methods: From November 2013 to March 2018, 458 patients (326 males, 132 females) were simultaneously examined using FA and/or SB and TP-PCR by detecting CGG repeat numbers in FMR1 gene and diagnosed as per American College of Medical Genetics guidelines., Results: The TP-PCR results showed high concordance with the FA and/or SB results for all three aspects (allele categorization, repeat number correlation, and zygosity concordance in female genetic carriers). TP-PCR detected CGG expansions ≥200 in all full mutation (FM) allele cases in male patients, as well as both the normal allele (NL) and FM allele in female carriers. In premutation (PM) allele carriers, the TP-PCR results were consistent with the FA and/or SB results. In terms of zygosity concordance in female genetic carriers, 12 NL cases detected by TP-PCR showed a merged peak consisting of two close heterozygous peaks; however, this issue was resolved using a 10-fold dilution., Conclusions: TP-PCR may serve as a reliable alternative method for FXS diagnosis.

Background: Chronological age estimation is a challenging marker in the field of forensic medicine. The current study aimed to investigate the accuracy of signal joint T-cell receptor rearrangement excision circles (sjTRECs) quantification and telomere length measurement as methods for estimating chronological age., Methods: Telomere length was estimated in the DNA derived from the buccal cells through estimating the telomeric restriction fragment (TRF) length using Telo TTAGGG Telomere Length Assay while the sjTRECs quantification was carried out on DNA isolated from the blood samples using qPCR., Results: The TRF length was shortened with increased age ( r = -0.722, p  < 0.001). The sjTRECs were also decreased with increased age ( r = -0.831, p  < 0.001). Stronger coefficient and lower standard error of the estimate was obtained when multiple regression analysis for age prediction based on the values of both methods was applied ( r = -0.876, p  < 0.001).

The ribosomal RNA (rDNA) sequence is the most abundant repetitive element in the budding yeast genome and forms a tandem cluster of ~100-200 copies. Cells frequently change their rDNA copy number, making rDNA the most unstable region in the budding yeast genome. The rDNA region experiences programmed replication fork arrest and subsequent formation of DNA double-strand breaks (DSBs), which are the main drivers of rDNA instability. The rDNA region offers a unique system to understand the mechanisms that respond to replication fork arrest as well as the mechanisms that regulate repeat instability. This chapter describes three methods to assess rDNA instability.

DNA double-strand break (DSB) end resection is an essential step for homologous recombination. It generates 3' single-stranded DNA needed for the loading of the strand exchange proteins and DNA damage checkpoint proteins. To study the mechanism of end resection in fission yeast, we apply a robust, quantitative and inducible assay. Resection is followed at a single per genome DSB synchronously generated by the tet-inducible I-PpoI endonuclease. An additional assay to follow resection involves recombination between two direct repeats by single-strand annealing (SSA), since SSA requires extensive resection to expose two single-strand repeats for annealing. The kinetics of resection and SSA repair are then measured using Southern blots.

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., (© 2020 John Wiley & Sons, Ltd.)

Subtelomere Y' elements get amplified by homologous recombination in sustaining the survival and division of the budding yeast Saccharomyces cerevisiae. However, current method for measurement of the subtelomere structures uses Southern blotting with labelled specific probes, which is laborious and time-consuming. By multiple sequence alignment analysis of all 19 subtelomere Y' elements across the 13 chromosomes of the sequenced S288C strain deposited in the yeast genome SGD database, we identified 12 consensus and relative longer fragments and 14 pairs of unique primers for real-time quantitative PCR analysis. With a PAC2 or ACT1 located near the centromere of chromosome V and VI as internal controls, these primers were applied to real-time quantitative PCR analysis, so the relative Y' element intensity normalised to that of wild type (WT) cells was calculated for subtelomere Y' element copy numbers across all different chromosomes using the formula: 2^[-((CT mutant Y' - CT mutant control ) - (CT WT Y' - CT WT control ))]. This novel quantitative subtelomere amplification assay across chromosomes by real-time PCR proves to be a much simpler and more sensitive way than the traditional Southern blotting method to analyse the Y' element recombination events in survivors derived from telomerase deficiency or recruitment failure., (© 2020 John Wiley & Sons, Ltd.)

Neutral-neutral 2-dimensional agarose gel electrophoresis enables the detection of replication intermediate structures in DNA. Here we describe how DNA from Escherichia coli cells can be purified to retain replication intermediates and then be separated by size and shape using two consecutive agarose gel electrophoresis protocols. The DNA structures present within a localized region can be visualized by a Southern blotting/radioactive hybridisation protocol.

A single cell can contain several thousand copies of the mitochondrial DNA genome or mtDNA. Tools for assessing mtDNA content are necessary for clinical and toxicological research, as mtDNA depletion is linked to genetic disease and drug toxicity. For instance, mtDNA depletion syndromes are typically fatal childhood disorders that are characterized by severe declines in mtDNA content in affected tissues. Mitochondrial toxicity and mtDNA depletion have also been reported in human immunodeficiency virus-infected patients treated with certain nucleoside reverse transcriptase inhibitors. Further, cell culture studies have demonstrated that exposure to oxidative stress stimulates mtDNA degradation. Here we outline a Southern blot and nonradioactive digoxigenin-labeled probe hybridization method to estimate mtDNA content in human genomic DNA samples. © 2019 by John Wiley & Sons, Inc., (© 2019 John Wiley & Sons, Inc.)

Ultraviolet rays induce interstrand and intrastrand DNA cross-links, usually thymine-thymine cyclobutane dimer (T-T) and thymine-thymine pyrimidine-pyrimidone (6-4) photoproduct (T (6-4) T). These DNA cross-links, if left unrepaired, increase the risk of these mutation being incorporated in the genetic material (i.e., DNA). Numerous studies have reported the mutagenic potential of above mentioned DNA adducts in prokaryotes, yeast and mammalian cells. Different techniques have been developed to identify such DNA adducts such as immuno-Southern blotting. This is a routinely used quantitative method to determine especially the amount of thymine dimers formed, following irradiation. In this chapter, the detailed methodology to identify thymine dimers formation is provided, using specific antibody against these adducts.

Accelerated cellular aging is known to play an important role in the etiology of phenotypes associated with developmental programming, such as cardiovascular disease and type 2 diabetes. Telomere length analysis is a powerful tool to quantify cellular aging. Here we describe a telomere length methodology, refined to quantify discrete telomere length fragments. We have shown this method to be more sensitive in detecting small changes in telomere length than the traditional average telomere length comparisons.

Here, we review the results of Southern blotting analyses of the FMR1 gene performed in our reference laboratory in Taiwan over a 15-year period. In total, 725 high-risk women with a family history of fragile X syndrome (FXS) or idiopathic intellectual disability, 3911 low-risk pregnant women without such family history, and prenatal diagnosis data for 32 foetuses from 24 carrier mothers were included. Only 2 carriers were in the low-risk group, which indicated a prevalence of 1 of 1955 women (95% confidence interval: 1/7156-1/539). A total of 100 carriers were found to be in the high-risk group, thus revealing a significantly higher frequency than the low-risk group (100/725 vs 2/3911, P<0.0001). Eight of the 14 foetuses that inherited the maternal mutant allele were verified to have a full mutation, with the smallest maternal pre-mutation allele carrying 56 CGG repeats. The overall findings confirmed that the carrier prevalence among low-risk women in Taiwan is significantly lower than that reported in western countries. Therefore, the most important step for preventing FXS in Taiwan would be to focus on high-risk women by promoting general awareness of this disease and spreading knowledge regarding the benefits of carrier screening and prenatal testing., (© 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Caldicellulosiruptor bescii is the most thermophilic cellulose degrader known and is of great interest because of its ability to degrade nonpretreated plant biomass. For biotechnological applications, an efficient genetic system is required to engineer it to convert plant biomass into desired products. To date, two different genetically tractable lineages of C. bescii strains have been generated. The first (JWCB005) is based on a random deletion within the pyrimidine biosynthesis genes pyrFA , and the second (MACB1018) is based on the targeted deletion of pyrE , making use of a kanamycin resistance marker. Importantly, an active insertion element, IS Cbe4 , was discovered in C. bescii when it disrupted the gene for lactate dehydrogenase ( ldh ) in strain JWCB018, constructed in the JWCB005 background. Additional instances of IS Cbe4 movement in other strains of this lineage are presented herein. These observations raise concerns about the genetic stability of such strains and their use as metabolic engineering platforms. In order to investigate genome stability in engineered strains of C. bescii from the two lineages, genome sequencing and Southern blot analyses were performed. The evidence presented shows a dramatic increase in the number of single nucleotide polymorphisms, insertions/deletions, and IS Cbe4 elements within the genome of JWCB005, leading to massive genome rearrangements in its daughter strain, JWCB018. Such dramatic effects were not evident in the newer MACB1018 lineage, indicating that JWCB005 and its daughter strains are not suitable for metabolic engineering purposes in C. bescii Furthermore, a facile approach for assessing genomic stability in C. bescii has been established. IMPORTANCE Caldicellulosiruptor bescii is a cellulolytic extremely thermophilic bacterium of great interest for metabolic engineering efforts geared toward lignocellulosic biofuel and bio-based chemical production. Genetic technology in C. bescii has led to the development of two uracil auxotrophic genetic background strains for metabolic engineering. We show that strains derived from the genetic background containing a random deletion in uracil biosynthesis genes ( pyrFA ) have a dramatic increase in the number of single nucleotide polymorphisms, insertions/deletions, and IS Cbe4 insertion elements in their genomes compared to the wild type. At least one daughter strain of this lineage also contains large-scale genome rearrangements that are flanked by these IS Cbe4 elements. In contrast, strains developed from the second background strain developed using a targeted deletion strategy of the uracil biosynthetic gene pyrE have a stable genome structure, making them preferable for future metabolic engineering studies., (Copyright © 2017 American Society for Microbiology.)

Human reproductive fitness depends upon telomere chemistry. Maternal age, meiotic nondisjunction error and telomere length of mother of trisomic child are someway associated. Reports exhibiting maternal inheritance of telomere length in Down syndrome child are very scanty. To investigate this, we collected peripheral blood from 170 mothers of Down syndrome child and 186 age matched mothers of euploid child with their newly born babies. Telomere length was measured by restriction digestion - southern blotting technique. Meiotic nondisjunction error was detected by STR genotyping. Subjects are classified by age (old >35 years and young ˂35 years) and by meiotic error (MI and MII). Linear regression was run to explore the age - telomere length relationship in each maternal groups. The study reveals that with age, telomere erodes in length. Old MII mothers carry the shortest (p˂0.001), control mothers have the longest telomere and MI lies in between. Babies from older mother have longer telomere (p˂0.001) moreover; telomeres are longer in Down syndrome babies than control babies (p˂0.001). To conclude, this study represents not only the relation between maternal aging and telomere length but also explore the maternal heritability of telomere length in families with Down syndrome child., (Copyright © 2017 Elsevier B.V. All rights reserved.)

An intronic GGGGCC hexanucleotide repeat expansion in the chromosome 9 open reading frame 72 (C9orf72) gene was considered as the most common cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia in Caucasian populations. Using repeat-primed polymerase chain reaction analysis and Southern blotting methods, we assessed the frequency and size of hexanucleotide repeat expansion in a cohort of 918 sporadic ALS (SALS) patients and 632 control individuals of Han Chinese origin. We identified 8 (0.87%) of the SALS patients and none of control individuals as carriers of C9orf72 expansions with 700-3500 repeats. A comprehensive neuropsychological battery was conducted on 4 expansion-positive ALS patients, where 3 patients were found to have cognitive impairment. All expansion-positive patients were genotyped for the previously reported 20 single-nucleotide polymorphism (SNP) risk haplotypes on chromosome 9p21. Among them, 13 SNP risk haplotypes were shared in all expansion carriers, suggesting a common founder from European ancestry. Further meta-analysis demonstrated that the intermediate expansion size with 24-30 repeats, rare in both patients and controls, were significantly associated with the risk for ALS. To our knowledge, this is the first study to identify a proportion of Chinese SALS patients carrying this pathologic expansion of up to ∼3500 repeats and to completely elaborate the 20-SNP risk haplotypes in Chinese expansion-positive patients, providing indispensable evidence for the origin, geographical range, and population prevalence of the C9orf72-associated ALS., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Contour-clamped homogenous electric field (CHEF) gel electrophoresis, a variant of Pulsed-field gel electrophoresis (PFGE), is a powerful technique for resolving large fragments of DNA (10 kb-9 Mb). CHEF has many applications including the physical mapping of chromosomes, artificial chromosomes, and sub-chromosomal DNA fragments, etc. Here, we describe the use of CHEF and two-dimensional gel electrophoresis to analyze rRNA gene methylation patterns within the two ~4 million base pair nucleolus organizer regions (NORs) of Arabidopsis thaliana. The method involves CHEF gel electrophoresis of agarose-embedded DNA following restriction endonuclease digestion to cut the NORs into large but resolvable segments, followed by digestion with methylation-sensitive restriction endonucleases and conventional (or CHEF) gel electrophoresis, in a second dimension. Resulting products are then detected by Southern blotting or PCR analyses capable of discriminating rRNA gene subtypes.

In the present study, a protocol for Agrobacterium tumefaciens -mediated transformation has been optimized for Woodfordia fruticosa (L.) Kurz. Precultured axenic leaf segments were co-cultivated with A. tumefaciens strain LBA4404 harboring the binary plasmid pCAMBIA1301 with β-glucuronidase ( uidA ) containing intron as the reporter gene and hygromycin phosphotransferase ( hpt ) as a selectable marker gene. After 3 days of co-cultivation, leaf segments were cultured on MS medium containing Thidiazuron (TDZ 4.54 μM) and Indole-3-acetic acid IAA (1.14 μM) + 20 mg/l hygromycin + 200 mg/l cefotaxime (PTSM 1 ) for 4 weeks (includes a single subculture onto the same medium at a 2 week interval). They were subsequently cultured for 3 weeks on MS medium containing Thidiazuron (TDZ 4.54 μM) and Indole-3-acetic acid IAA (1.14 μM) + 25 mg/l hygromycin + 100 mg/l cefotaxime (PTSM 2 ) medium for further development and shoot elongation. The hygromycin resistant shoots were rooted on a rooting medium (PTRM) containing half strength MS medium + 4.90 μM IBA + 25 mg/l hygromycin. A highest transformation efficiency of 44.5% with a mean number of 2.6 transgenic shoots per explant was achieved. Successful transformation was confirmed by the histochemical GUS activity of the regenerated shoots, PCR and RT-PCR analysis using respective primers. Southern blot analysis revealed that the hpt gene integrated into the genome of transgenic W. fruticosa . Establishment of genetic transformation protocol may facilitate the improvement of this medicinal plant in terms of enhancement of secondary metabolites.

Histomonas meleagridis, a protozoan parasite that can infect gallinaceous birds, affects mainly the liver and caeca of infected birds. As a consequence of the recent ban of chemotherapeuticals in Europe and the USA, histomonosis gained somewhat more attention due to its re-emergence and the fact that there is no effective treatment available. Therefore, special attention is now also given towards the diagnosis and the control of the disease. In the actual study we report the development of highly specific and sensitive real-time PCR methods for detection and quantification of the parasite, based on two protein coding genes, Fe-hydrogenase (FeHYD) and rpb1. Both genes seem to be in a single copy in H. meleagridis as shown by southern blotting and absolute quantification using real-time PCRs on samples containing a known amount of the parasite. The real-time PCR assays based on FeHYD and rpb1 genes were found to be an efficient method for the quantification and detection of H. meleagridis in in vitro grown cultures, tissues of infected birds and in faecal samples. Both real-time PCRs were able to detect up to a single cell in in vitro cultures of H. meleagridis and in fecal samples spiked with H. meleagridis. Finally, qPCR assays were shown to be highly specific for H. meleagridis as samples containing either of the two H. meleagridis genotypes were positive, whereas samples containing other protozoa such as Tetratrichomonas gallinarum, Trichomonas gallinae, Simplicimonas sp., Tritrichomonas sp., Parahistomonas wenrichi, Dientamoebidae sp. and Blastocystis sp. were all negative., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Cre-inducible mouse models are often utilized for the spatial and temporal expression of oncogenes. With the wide number of Cre recombinase lines available, inducible transgenesis represents a tractable approach to achieve discrete oncogene expression. Here, we describe a protocol for targeting Cre-inducible genes to the ubiquitously expressed ROSA26 locus. Gene targeting provides several advantages over standard transgenic techniques, including a known site of integration and previously characterized pattern of expression. Historically, an inherent instability of ROSA26 targeting vectors has hampered the efficiency of developing ROSA26 knock-in lines. In this protocol, we provide individual steps for utilizing Gateway recombination for cloning as well as detailed instructions for screening targeted ES cell clones. By following this protocol, one can achieve germline transmission of a ROSA26 knock-in line within several months., (Copyright © 2015 John Wiley & Sons, Inc.)

An intronic G(4)C(2) hexanucleotide repeat expansion in C9ORF72 is a major cause of amyotrophic lateral sclerosis and frontotemporal lobar degeneration. Several mechanisms including RNA toxicity, repeat-associated non-AUG translation mediated dipeptide protein aggregates, and haploinsufficiency of C9orf72 have been implicated in the molecular pathogenesis of this disorder. The aims of this study were to compare the use of two different Southern blot probes for detection of repeat expansions in an amyotrophic lateral sclerosis and frontotemporal lobar degeneration pathological cohort and to determine the levels of C9orf72 transcript variants and protein isoforms in patients versus control subjects. Our Southern blot studies identified smaller repeat expansions (250-1800 bp) that were only detectable with the flanking probe highlighting the potential for divergent results using different Southern blotting protocols that could complicate genotype-phenotype correlation studies. Further, we characterize a new C9orf72 antibody and show for the first time decreased C9orf72 protein levels in the frontal cortex from patients with a pathological hexanucleotide repeat expansion. These data suggest that a reduction in C9orf72 protein may be a consequence of the disease., (Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.)

Crambe abyssinica is a dedicated oilseed crop suitable for production of industrial feedstocks. Genetic modification of crambe has progressed substantially in the last few years, but the transformation efficiency needs to be further improved. Meanwhile, developing a reliable molecular system including Southern blot and qRT-PCR analyses is desired for effectively evaluating transgenic lines and gene expression levels of both endogenous and transgenes. In this study, we have developed an efficient transformation protocol with hygromycin as the selective agent for crambe transformation. In the regeneration test, addition of hygromycin at concentration of 5 mg L(-1) resulted in 18% of shoot regeneration using crambe hypocotyls as explants, while no regeneration occurred when the hygromycin concentration reached 10 mg L(-1). Based on this result, the hygromycin concentration up to 10 mg L(-1) was used in the subsequent transformations. The results showed that the transformation efficiency under constant low selection pressure (H3-H3) was similar to that under higher selection pressure first, followed by transfer to lower selection pressure (H10-H3). The PCR, Southern blot and fatty acid composition analyses confirmed the integration of transgenes in the crambe genome. We have also optimized the Southern and qRT-PCR methods for future studies on crambe or related species. For Southern blot analysis on crambe, more than 50 μg DNA is required for a clear band. The choice of enzymes for DNA digestion was not rigid for confirmation of the T-DNA integration, while for determining the copy number of transgenes, suitable enzymes should be chosen. Increasing the enzyme concentration could improve the digestion and 20 μl enzyme was recomended for a complete digestion of up to 80 μg crambe DNA. For qRT-PCR analysis, around 20 days after flowering was observed to be the suitable sampling time for expresseion analysis of genes invovled in the seed oil biosynthesis.

The purpose of this protocol is to detect specific DNA sequences in a complex sample by hybridization to a labeled probe., (Copyright © 2013 Elsevier Inc. All rights reserved.)