DNA Isolation Research Articles

Genetic Diversity of Cultured Rainbow Trout (Oncorhynchus mykiss) Populations in Turkey Based on Mitochondrial DNA Cytochrome b (cyt-b) Sequence Analysis

This study aimed to investigate the genetic diversity and structure of rainbow trout (Oncorhynchus mykiss) cultured populations in different provinces of Turkey, based on the cytochrome b (cyt-b) gene region of mitochondrial DNA (mtDNA). Tissue samples were collected from a total of 98 fish (seven fish per province) across 14 provinces, followed by DNA isolation. The mtDNA cyt-b gene region (754 bp) was amplified using PCR. Genetic diversity indices, genetic structure, and phylogenetic analyses were calculated from the cyt-b gene sequence data. The average nucleotide frequencies of the four nucleotides cytosine (C), thymine (T), adenine (A), and guanine (G) were found to be 26.2%, 24.9%, 26.2%, and 22.8%, respectively, with a higher A + T content (51%) compared to G + C content (49%). A total of 645 polymorphic loci were identified across the 14 populations, and 50 distinct haplotypes were defined. Haplotypic diversity ranged from 0.91 to 0.48, while nucleotide diversity (π) varied between 0.26 and 0.00. The highest genetic diversity was observed in the Tokat and Van populations, whereas the lowest was recorded in the Elazığ and Kahramanmaraş populations. AMOVA analyses revealed that 71.1% of the genetic diversity was found between populations, while 28.9% was found within populations. Pairwise FST values ranged from 0.38 to 0.99, with an average FST of 0.71. Phylogenetic analyses indicated that the populations clustered into two main groups, with further sub-groupings within these clusters. Notably, the Kahramanmaraş and Elazığ populations were found to be significantly differentiated from other populations. In conclusion, despite rainbow trout not being a native species in Turkey, the findings of this study indicate that the genetic diversity of the populations is at a good level. However, in order to preserve the genetic diversity of cultured rainbow trout populations and to increase effective population size, it is recommended that breeders engage in fry exchanges and import new rainbow trout specimens to enhance genetic diversity.

Investigation of the Relationship Between ACTN3 rs1815739 Polymorphism and Openbite Cases: A Prospective Study.

The aim of this study was to examine whether ACTN3 rs1815739 polymorphism, which causes the deficiency of the alpha-actinin-3 muscle protein, is related to the formation of open bite malocclusion. Fifty-eight participants (18.5 ± 3.6 years old) with anterior open bite (n = 29) and normal overbite (n = 29) who presented to Marmara University, Department of Orthodontics for treatment were included in the study. Initial cephalometric radiographs were used for the diagnosis of malocclusion. The case group was divided into three subgroups according to degree of open bite. For DNA isolation, oral epithelial cells were collected with buccal swabs (Van Allen Way, Carlsbad, USA), and the real-time PCR method was used for the genotyping of all polymorphisms. The results were statistically analysed, and the threshold for statistical significance was set at p < 0.05. The frequencies of RR, RX and XX genotypes of ACTN3 rs1815739 polymorphism were found as 6 (20.7%), 14 (48.3%) and 9 (31.0%) in the control group and 8 (8%), 9 (31.0%) and 12 (41.4%) in the case group, respectively. There was no statistically significant difference between the groups in terms of the presence of the examined polymorphism (p > 0.05). However, the intra-group evaluation of case group revealed a significant difference in the prevalence of XX genotype (83.3%) for the subgroup with an open bite of -5 mm or above. Although no significant difference was observed between the case and control groups, a possible association was identified between ACTN3 rs1815739 polymorphism and an increased severity of open bite malocclusion.

First Report of the Gene Mutations Associated with Permethrin Resistance in Head Lice (Pediculus humanus capitis De Geer, 1767) from Primary School Children in Istanbul (Türkiye) and Nagarkot (Nepal).

Head lice infestation (HLI), caused by Pediculus humanus capitis De Geer, 1767, has long been a common global problem of school children. Permethrin is an old pyrethroid derivative that has been used commonly for its treatment, and it exerts its activity over the voltage-sensitive calcium channels (VSCC) of the lice. There has been a growing list of persistent HLI cases lately in the world among patients using permethrin, and knockdown resistance (kdr)-related point mutations on VSCC have been identified and reported from those resistant lice samples. The aim of this study was to investigate the gene mutations associated with permethrin resistance in head lice collected from primary school children in Istanbul (Türkiye) and Nagarkot (Nepal) for the first time. A total of 192 P. h. capitis adults were collected from school children aged 6-12 years in two cities (96 lice each). Following DNA isolation, the fragment of the VSCC a-subunit gene, which contained the possible mutation sites ((kdr-like M815I (ATG > ATT), kdr T917I (ACA > ATA), and kdr-like L920F (CTT > TTT)), was amplified in each louse by PCR, and the PCR products were sequenced and aligned, followed by frequency calculations for alleles, genotypes, and haplotypes. Using nucleic acid sequence analysis, it was revealed that M815I, T917I, or L920F mutations were present on the VSCC genes in the lice samples from both Türkiye and Nepal. In addition, genotypic analyses indicated the presence of all three mutations in the lice samples from Türkiye, while the T917I mutation was detected in none of the lice collected in Nepal. This is the first report of gene mutations associated with permethrin resistance in head lice collected from a group of primary school children in the largest city of Türkiye (Istanbul) and Nagarkot. High mutation rates were identified in the lice, especially those from Istanbul, which is concordant with our previous unpublished study, in which almost 60% of the examined lice of the school children (in the same school selected in this study) remained alive despite long-term exposure to permethrin in the laboratory. These initial results show that gene mutations associated with permethrin resistance are common in lice samples in Istanbul and Nagarkot, which may suggest the current need for the selection of new pediculicidal agents in HLI treatment.

Pilot progress in DNA isolation and amplification from the material stored at the LWS herbarium

The isolation of DNA from the herbarium specimens deposited at the LWS herbarium (State Museum of Natural History of the NAS of Ukraine, Lviv, Ukraine) has been tested using the column-based protocol. The isolated DNA has been amplified using different nuclear and plastid primers. The yield of obtained total DNA showed no significant dependence from the year of collection and plant family of studied specimens. In general, the obtained DNA of LWS specimens had medium yield (mean – 56.47 ng/µL) but relatively low purity (mean 260/230 value – 0.85 units and mean 260/280 value – 1.66 units). The success of DNA amplification for old herbarium material varied from 12.5 % to 91.1 % depending on applied primers. The trnL P6 Loop primers demonstrated the best performance (91.1 % successful amplification), but due to short resulted DNA fragments, it was not possible to purify the product for further processing. UniPlant primers performed the worst, and only 12.5 % of samples taken from the LWS herbarium (excluding controls) were successfully amplified. In general, nuclear primers, except for UniPlant, demonstrated a better success rate (mean – 31.5 %) during the work with samples taken from the LWS herbarium. Meanwhile, the plastid primers, except for trnL P6 Loop, showed slightly lower amplification success (mean – 26.8 %).

Genetic Polymorphism of TAP1, TAP2 and Tapasin Genes in Exotic and Indian Native Chicken Breeds

Background: Indigenous chicken breeds have shown resilience to common poultry diseases due to their genetic background. Research has highlighted the role of certain genes in the immune response and disease resistance in poultry. Among these, Tapasin and Transporter associated with antigen processing (TAP) genes have been identified as crucial players in the immune surveillance of avian species. However, very diminutive information is available on the genetic polymorphism of TAP1, TAP2 and Tapasin genes in Indian native chicken breeds Methods: A total of 362 birds from three distinct breeds, namely Brown Nicobari (female: 55, male:64), Ghagus (female: 58; male:60)and Dahlem Red (female: 57; male:68), were used in the study. DNA isolation was performed, followed by PCR amplification using various primers from the three genes. Single-Strand Confirmation Polymorphism (SSCP) analysis was carried out on the PCR products to identify any potential single nucleotide polymorphisms (SNPs). These SNPs were then confirmed through sequencing. Result: The study has confirmed that these genes are polymorphic in all the three studied chicken breeds. In the exon 7 of TAP1 gene, a polymorphic SNP at nucleotide position 3698 (GAG 336 AAG) was identified. A non-synonymous substitution at nucleotide position 4209 (GCA 371 ACA) in the exon8 of TAP1 gene was observed. In exon 6 of TAP2 gene, three variations were observed and of which the variation at nucleotide position 2658 (GAC 446 AAC) is non-synonymous. In exon 7 and 8 of TAP2 gene, a total of three SNPs were observed of which a non-synonymous substitution was observed at nucleotide position 3125(AGG 579AAG) in exon 7. In exon 5 of Tapasin gene, three non-synonymous substitution at nucleotide 3226 (ACG 293ATG), 3292 (CAC 315 CGC) and 3466 (ATT 373ACT) were identified.

First Report of Maize Ear Rot Caused by Fusarium boothii in Serbia.

Fusarium graminearum species complex (FGSC) includes at least fifteen species which are some of the most significant fungi that infect maize in temperate areas (Sarver et al. 2011). Agroecological conditions in Serbia are suitable for the development of infection by members of FGSC and therefore during the period of 1993-2010, maize samples collected from northern Serbia (46°5'55" N, 19°39'47" E) showed typical symptoms of gibberella ear rot. Twenty isolates were selected for study for FGSC identification. Appearance of colonies and macroconidia on potato dextrose agar (PDA) of all isolates (average 37.75-45.35×4.35-5.35 µm, No 50) were consistent with descriptions of F. graminearum (O'Donnell et al. 2004). Monosporal isolates were grown on PDA and used for molecular analyses. DNA isolation was performed using DNeasy Plant Mini Kit. Identification of FGSC was performed on the basis of the TEF-1α gene amplified with primers ef1/ef2 (Geiser et al. 2004) and sequenced in both directions. In four selected isolates two additional genomic regions (histone H3 and β-tubulin) were further analyzed using primers H3-1a/H3-1b and T1/T22, respectively (O'Donnell et al. 2000, Glass and Donaldson 1995). Nucleotide sequences of TEF-1α, β-tubulin, histone H3 have been deposited in GenBank under accession numbers: isolate 914 (MF974400, MG063784, MF999140), 1495 (MF974405, MG063789, MF999145), 2812 (MF974408, MG063792, MF999148), 2822 (MF974409, MG063793, MF999149), respectively. Isolates were molecularly identified using BLAST tool from NCBI and with phylogenetic analyses (Maximum Parsimony method with 1000 bootstrap replications on concatenated sequences of all three genes-TEF-1α, β-tubulin and histone H3) using MEGA 11 software package (Tamura et al. 2011). In BLAST analyses isolate 2822 shared 100% nucleotide identity with reference isolate Fusarium boothii NRRL26916 (GQ915470) originating from Central America based on histone H3 gene. Isolates 914, 1495 and 2812 shared 99% to 100% nucleotide identity with F. graminearum isolates. Phylogenetic analyses showed that all equally parsimonious trees made of 32 selected sequences of species within FGSC available in GenBank database and our four isolates, rooted with external outgroup species F. pseudograminearum, grouped three isolates (914, 1495, 2812) with F. graminearum isolates (NRRL28336, 29169, 28439) and one isolate (2822) grouped with F. boothii isolates (NRRL 29020, 26916). Pathogenicity of isolates was confirmed using the method of Reid et al. (1996). Artificial inoculation of maize ears was performed on the third day after silking by injecting 2 ml of a conidial suspension with a concentration of 1x105 conidia/ml into the silk channel. The same procedure was applied to control plants, using sterile water instead of inoculum. After three weeks, symptoms of Gibberella ear rot appeared and the pathogen was again successfully isolated. The fungus was identical to original isolates, thus completing Koch's postulates. Control plants did not show any symptoms. Based on available literature, this is the first report of species F. boothii in Serbia. Future research should be focused on determining distribution, aggressiveness, synthesis of mycotoxins, as well as damage caused by F. boothii on grain yield and quality compared to F. graminearum. This will contribute to a better understanding of biodiversity of FGSC, which may lead to a successful strategy for controlling these harmful pathogens.

ENHANCING YIELD OF LOW COPY NUMBER PLANT BASED VECTOR FROM E.COLI CELLS FOR GENE CLONING

Plasmid DNA is an important role play for gene cloning and gene expression analysis in E.coli cells. But some of the plasmids are very low copy number so its isolation is also difficult due to its low copy number. So in this study we used different types of media (broths) with more volume, area, specific growth temperature, time of incubation and quality of inoculum. We used five different types of broth for enhancing the yield of low copy number plasmids such as LB, LB24, SB, SOC and 2xYT media. In these five media the differences is low and high concentration of yeast extract and tryptone and in SOC media some different salts and glucose are additional. Comparison of these five media for enhancing the yield of low copy number plasmid DNA, SOC broth optical density is more comparative to other media. In SOC media plasmid culture OD and isolated plasmid DNA concentration both are directly proportional to each other. For enhancing the yield of low copy number plasmid DNA in E.coli cells these following factors are very important role play, quality of inoculum,size of inoculum specific incubation temperature, area of shaking vessel, incubation time, required concentration of antibiotics, and also concentration of tryptone, yeast extract and additional salts and glucose. One another important factor for enhancing the yield of low copy number plasmid used manual method (alkaline lysis method) for isolation of low copy number plasmid DNA and its purification also effects the yield of plasmid DNA.

Multidimensional Approach of Genotype and Phenotype in Stroke Etiology: The MAGPIE Study.

Stroke is a leading cause of mortality and morbidity in Bangladesh. It is estimated that genetic determinants account for around 40%-60% of its etiology, similar to environmental factors. This study aimed to provide a better understanding of the genetic, environmental, and clinical risk factors in stroke patients from Bangladesh. The MAGPIE (Multidimensional Approach of Genotype and Phenotype In Stroke Etiology) study is a population-based case-control study that will allow a hypothesis-free genome-wide association study (GWAS) to identify genetic risk factors associated with adult stroke (age ≥ 18 years) in Bangladesh. This study will collect detailed phenotypic data as well as blood samples from stroke patients and control subjects. High-molecular-weight genomic DNA will be isolated and archived using Qiagen DNA isolation kits. We will utilize SPSS v28.0, vR-4.3.2 and gPLINK v2.0 software to analyse the study variables, as appropriate. Further, appropriate statistical tests will be applied to test the significance level between study groups. As applicable, data will be presented in tables and graphs, such as Manhattan plots and Quantile-Quantile (QQ) plots. A p < 0.05 will be considered as statistical significance. This will be the first large-scale carefully phenotyped biobank of Bangladeshi stroke patients which will enable a GWAS enabling an understanding of the association between gene-phenotype risk factors which has the potential to revise and refine national stroke guidelines.

Combined systems of polymerase chain reaction and a time-resolved fluorescence immunoassay or membrane immunochromatography for quantitative determination of Salmonella enterica bacterial DNA

Four model bioanalytical systems specific for Salmonella enterica have been developed and studied, in which a polymerase chain reaction (PCR) produced a DNA amplicon containing biotin and fluorescein residues. This enabled to immobilize the amplicon on a functionalized solid phase and to label it biospecifically with europium chelate in microplates or gold nanoparticles on a chromatographic membrane. Quantitative detection of the modified DNA was carried out in immunoassay systems by measuring the Eu3+ time-resolved fluorescence (dissociation-enhanced lanthanide fluorescence immunoassay, DELFIA) or by photometry of the colored zone on the chromatographic strip (LFA). Three pairs of primers were developed and examined to obtain selected fragments of the invA gene, which is present in the genomes of all pathogenic Salmonella enterica. The fragments proved to be suitable for the test systems. In the microplate DELFIA system, the concentration range of DNA amplicon quantification was found to be 0.01–10.0 nM, and a detection limit was 2 pM. The limit of DNA visual detection in LFA was 0.05 nM. The possibility of testing the amplicons without additional isolation of pure DNA from the reaction mixture was demonstrated. The high specificity of the developed bioanalytical systems for the detection of various Salmonella enterica serotypes was demonstrated.

A Sensitive, Specific and Fast Electrochemical‐based Nanobiosensor Diagnostic for Xanthomonas albilineans, the Cause of Sugarcane Leaf Scald Disease

AbstractLeaf scald (LS) caused by Xanthomonas albilineans (Xalb), is a major bacterial disease of sugarcane. The unreliable symptom expressions make traditional visual detection challenging. The molecular methods of detection require expensive equipment, labor‐intensive, and time‐consuming. This study proposes a novel electrochemical (EC)‐approach, that is relatively easy to use and less expensive to detect Xalb DNA in LS‐infected sugarcane leaves, meristematic tissue, and xylem sap samples. This method involves three key steps: i) DNA isolation from sugarcane samples via boiling lysis; ii) magnetic purification of target sequences from the lysate using magnetic bead‐bound capture probes; and iii) EC detection of the target DNA. The method shows excellent detection sensitivity (10 cells µL−1), reproducibility (Standard deviation, SD &lt;5%, for n = 3), and a wide linear dynamic range (1 nM–1 fM or 106–10° copies µL−1, r = 0.99). The EC assay has a strong negative correlation with quantitative polymerase chain reaction (qPCR) results (r = −0.95–0.97, n = 24, p &lt; 0.001), and weak or no correlation with the varietal resistance ratings. This EC‐based assay can be a commercially viable alternative, providing a DNA isolation/purification‐free solution, and can potentially be adapted into a handheld device for on‐farm detection and quantification of the LS‐causing pathogen.

Association between KRAS, NRAS, and BRAF mutations and tumor localization in colorectal cancer patients in BSMMU

Background: Colorectal cancer (CRC) is a common malignancy with significant genetic heterogeneity. Mutations in proto-oncogenes such as KRAS, NRAS, and BRAF play a pivotal role in CRC development, impacting prognosis and treatment. This study aims to correlate mutations in these genes with tumor localization in both primary and metastatic CRC in the Bangabandhu Sheikh Mujib medical university (BSMMU) cohort. Methods: This prospective cross-sectional study was conducted between July 2023 and June 2024 at BSMMU. A total of 30 CRC patients, confirmed via histopathology, were included. Purposive sampling was used to select patients. Tumor tissue samples were collected and analyzed for KRAS, NRAS, and BRAF mutations using DNA isolation, PCR amplification, and sequencing techniques. Results: Among the 30 patients, the majority were male (66.7%) with a mean age of 50.4 years. KRAS mutations were found in 5 patients (16.7%), while no mutations in NRAS or BRAF were detected. Rectal cancer was the most frequent tumor location (36.7%), followed by hepatic and splenic flexure (16.7% each). No significant correlation was observed between KRAS mutations and tumor localization​. Conclusions: There was no statistically significant correlation between KRAS, NRAS, and BRAF mutations and tumor localization in the BSMMU CRC patient cohort. The study highlights the need for larger sample sizes to better understand the genetic landscape of CRC in Bangladesh​. Small sample size may limit the ability to detect significant associations. Further large-scale studies could offer more conclusive insights.

The Prospect of Gene Exploitation through Soil Metagenomics

The quantity and diversity of the microbial community in soil make it possibly the most difficult of all the natural ecosystems. It is thought to be challenging to culture up to 99% of the microorganisms in a given environment. The intricacy of microbial variety is impacted by numerous interconnected factors, including as soil structure, water content, biotic activity, pH, and fluctuations in climate. Environmental DNA isolation and purification are often the first steps in the soil metagenomic analysis process. Creating genomic DNA libraries and then using them for high-throughput sequencing or library screening are the main steps in the application of metagenomics. These genomic sequences are currently being used to advance our knowledge of the ecology and physiology of these bacteria as well as for new biotechnological and medicinal applications. To completely comprehend the intricacies involved in the operation of microbial communities and the interactions between different microorganisms within specific niches, metagenomic sequences are employed. This study focuses on the latest advancements in biotechnological approaches and procedures for identifying novel genes from uncultured microorganisms and intricate microbial habitats.

Rapid Detection of Listeria monocytogenes In Chicken Meat By Real-time PCR without Culture Enrichment

Foodborne pathogens can easily contaminate chicken meat due to its high nutritional content, and these pathogens can infect humans. One of the most important pathogens contaminating chicken meat and causing severe public health problems is Listeria monocytogenes, which would be responsible for Listeriosis. Therefore, rapid and sensitive detection of L. monocytogenes in chicken meat samples is of great significance. In the current study, the presence of L. monocytogenes in chicken meat samples collected from several markets in Erzurum was detected by comparing two different DNA isolation methods with the Real-time PCR. As a result of the analyses, it was determined that 34% of the chicken meat samples collected were positive for L. monocytogenes in both two methods. According to the comparison analyses of the Bland-Altman method, no significant difference was found between the thermal lysis method and the DNA isolation method by commercial kit. As a result of this study, it has been shown that the thermal lysis method can be successfully applied for the detection of foodborne pathogens in chicken meat when evaluated in terms of workload and cost. The current study is the first report on the comparison of thermal lysis method and DNA isolation by commercial kit in the detection of L. monocytogenes from chicken meat by Real-time PCR.

REASSURED Test System for Food Control—Preparation of LAMP Reaction Mixtures for In-Field Identification of Plant and Animal Species

The potential of loop-mediated isothermal amplification (LAMP) assays for species identification in medical diagnostics, food safety, and authentication is indisputable. The challenge in commercialization of such DNA-based rapid test methods for resource-limited settings is the on-site availability of the required reagents and an intuitive read-out system. In this work, reaction mixtures for LAMP assays for the detection of animal (plaice) and plant food (safflower) were lyophilized and stored at room temperature for up to 24 weeks, demonstrating that refrigeration of reagents after lyophilization is not absolutely necessary. During this period, reaction mixtures were stable and the polymerase showed no loss of activity. In addition, mobile testing, including DNA isolation, using the lyophilized LAMP reaction mixtures and a handheld fluorescence detection read-out system (Doctor Vida Pocket test) was successfully performed outside of the laboratory environment in less than 40 min using a proposed standard operation procedure. The results highlight that the use of the lyophilized LAMP reaction for food control purposes has the potential to meet the WHO-proposed REASSURED criteria.

Novel PCR-Based Technology for the Detection of Sunflower in Edible and Used Cooking Oils.

Reliable detection of sunflower (Helianthus annuus) in edible and used cooking oil (UCO) is crucial for the sustainable production of food and biodiesel. In this study, a variety of sunflower oils (crude, cold pressed, extra virgin, refined, and UCO) were examined using different methods of DNA extraction and PCR amplification to develop an efficient technology for the identification of sunflower in oils. DNA extraction kits such as NucleoSpin Food, DNeasy mericon Food, and Olive Oil DNA Isolation as well as modified CTAB method were found to be able to isolate amplifiable genomic DNA from highly processed oils. Novel uniplex, double, and nested PCR systems targeting the sunflower-specific helianthinin gene were developed for efficient identification of sunflower. New sunflower DNA markers were revealed by uniplex PCRs. The combination of modified CTAB and nested PCR was demonstrated as a reliable, rapid, and cost-effective technology for detecting traces of sunflower in 700 μL of highly processed oil, including refined and used cooking oil. The study will contribute to both the food industry and the energy sector as developed methods can be used for oil authenticity testing in food and biodiesel production.

Extraction of Genomic DNA from Different Plant Tissues through Phenol-chloroform Method

Background: DNA extraction is a fundamental technique in molecular biology that involves isolating DNA from biological samples for various downstream applications, including PCR and sequencing. Phenol-chloroform DNA extraction is an alternate method of the CTAB method for genomic DNA extraction. This method typically yields more DNA than the CTAB with high purity. It is useful for high molecular weight/long fragment DNA determinations. This study aims to extract DNA from plant tissues using the phenol-chloroform method and compare the response of different plant leaves to this method. Methods: In this study, we provide a step-by-step guide to DNA extraction from five different plant species, including Ficus capensis (Cape fig or Cape banyan), Ficus exaspirata (Forest Sandpaper), Mangifera indica (Mango), Gmelina arborea (Gamhar), and Bauhinia purpurea (Butterfly tree). The DNA isolation protocol involved several steps, such as grinding the plant tissue, adding NaCl solution, using a chloroform-isoamyl alcohol mixture to extract DNA, and further purification with phenol-chloroform-isoamyl alcohol. Isopropanol was used to precipitate the DNA, and ethanol was used to wash the DNA pellet to remove any remaining contaminants. Finally, the DNA pellet was dissolved in TE buffer for storage. Results: The results showed the successful implementation of the phenol chloroform method for isolating DNA from selected plant tissues. Overall, this study provides a comprehensive protocol for DNA extraction from plant tissue using readily available laboratory reagents and equipment. Conclusion: The protocol can be modified to accommodate different plant species and sample types and can facilitate further research in plant genetics and biotechnology.

Comparison of DNA isolation methods for GMO detection from biscuit samples

The global increase in genetically modified organism (GMO) content in feed and food products has necessitated the development of precise detection methods to differentiate between biotechnologically derived foods and those without GMOs. Despite the various regulations in different countries, an internationally consistent approach to labeling GMO products is needed. For this reason, there is a widespread need to develop effective GMO detection methods to provide reliable and transparent food safety to consumers. The first experimental step in creating accurate and reliable detection methods for GMOs is effective DNA isolation. Determining DNA isolation methods specific to different processing levels of foods is very important. This study was aimed to compare different DNA extraction methods in biscuit samples. For this reason, DNA from different biscuit samples was isolated using three different protocols (CTAB-PVP, modified CTAB-1, and modified CTAB-2). DNA samples extracted from biscuits, were evaluated regarding DNA concentration and purity. According to the results obtained, the CTAB-2 procedure generally showed the best performance in terms of both DNA amount and purity rates for biscuit samples.

Synthetic minichromosomes in plants: past, present, and promise.

The status of engineered mini-chromosomes/artificial chromosomes/synthetic chromosomes in plants is summarized. Their promise is that they provide a means to accumulate foreign genes on an independent entity other than the normal chromosomes, which would facilitate stacking of novel traits in a way that would not be linked to endogenous genes and that would facilitate transfer between lines. Centromeres in plants are epigenetic, and therefore the isolation of DNA underlying centromeres and reintroduction into plant cells will not establish a functional kinetochore, which obviates this approach for in vitro assembly of plant artificial chromosomes. This issue was bypassed by using telomere-mediated chromosomal truncation to produce mini-chromosomes with little more than an endogenous centromere that could in turn be used as a foundation to build synthetic chromosomes. Site-specific recombinases and various iterations of CRISPR-Cas9 editing provide many tools for the development and re-engineering of synthetic chromosomes.

An improved DNA extraction method in okra for rapid PCR detection of Okra enation leaf curl virus from diverse Indian regions.

The extraction of DNA from okra (Abelmoschus esculentus) is challenging due to its high mucilage and polysaccharide content, which can hinder both the yield and quality of DNA.In this study, an improved DNA isolation method is described incorporating a key modification being the use of solution I (1 M NaCl and 2% Sarcosyl) as a pre-treatment before applying the CTAB buffer, resulting in high-purity genomic DNA in just 1h and 45min., making it suitable for handling large sample sizes due to its rapid processing capabilities. This enhanced DNA extraction method was crucial for the accurate and rapid molecular detection of Okra enation leaf curl virus (OELCuV), a monopartitebegomovirus that has spread across various regions of India. Transmitted by the whitefly (Bemisia tabaci), OELCuV causes leaf curling, enations, and stunted growth in okra, leading to significant yield losses. The surveys conducted during the 2020-21 and 2021-22 sowing seasons revealed disease incidence ranging from 14.03 to 67.57%. The extracted DNA via the improved DNA extraction method enhanced the speed of PCR based molecular identification of OELCuV, using virus-specific coat protein primers. The amplified CP genes were cloned and sequenced to study the CP gene based diversity among OELCuV isolates from different states of India. The CP gene nucleotide identity among the studied OELCuV isolates ranged from 95.57 to 99.27%, while comparison with previously reported Indian OELCuV CP sequences, the nucleotide identity ranged from 89.35 to 98.83%.The successful application of this optimized DNA extraction method sped up the detection process but also holds promise for broader use in the molecular study of okra and other mucilaginous crops, particularly in the rapid and reliable identification ofbegomoviruses. The optimized DNA extraction method significantly accelerated the detection of OELCuV, demonstrating its efficiency and reliability. This method shows strong potential for broader applications in the molecular study of okra and other mucilaginous crops, making it a valuable tool for future research and disease management.

Abstract B073: Leveraging micronuclei DNA from erythrocytes for early detection of hepatocellular carcinoma

Abstract Background: Hepatocellular carcinoma (HCC) is the most common form of liver cancer and accounts for ∼90% of cases globally. Detection of early-stage HCC is vital as it allows for potentially curative treatment, yet remains a significant challenge. Micronuclei (MN) are a hallmark of genomic instability. An increased frequency of MN is commonly observed in tumor cells as well as other somatic cells, including erythrocytes, in cancer patients. Here, we have established a technique (WO2021/228246 A1), for the isolation and analysis of micronuclei DNA (MN-DNA) in erythrocytes from peripheral blood. Significant changes in read densities at specific genomic locations within MN-DNA were identified in patients, termed as tumor- associated MN-DNA (taMN-DNA) features. This study evaluates the potential of these taMN- DNA features for early-stage HCC detection across human and murine model. Methods: To explore the potential of MN-DNA in cancer detection, we first collected 1-2 mL of whole blood from HDs (N = 53) and HCC patients (N = 53). MN-DNA was isolated and purified from erythrocytes, followed by whole-genome sequencing. Participants were randomly divided into training, test cohorts in an 8:2 ratio. We applied machine learning algorithms to leverage these features for cancer detection in the human model. Additionally, to investigate the formation of taMN-DNA features, we employed a well-established mouse model that mimics HCC development. Mice were injected with the genotoxic agent diethylnitrosamine (DEN) 2 weeks after birth, and developed malignant macroscopic liver nodules at approximately 40 weeks of age. Results: In the human cohort, we enrolled 106 participants, with more than half of the HCC cases were diagnosed at early-stage (stage 0-II). The cancer detection model utilizing taMN- DNA features achieved an overall accuracy of 86.3%, with a sensitivity of 81.8% and specificity of 90.9%. For early-stage, the model demonstrated sensitivities of 54.5% at a specificity of 90.9%. In the DEN-induced HCC mouse model, unsupervised hierarchical clustering based on these selected taMN-DNA features clearly separated WT and tumor mice into two distinct groups. Notably, mice that were not tumorigenic at 36 weeks post-treatment in the DEN-treated group fell into the WT group when classified by the same taMN-DNA features, indicating that the formation of taMN-DNA signatures is associated with the presence of tumors specifically. Conclusions: This pilot study highlights the potential of MN-DNA as a promising tool for early HCC detection. Leveraging these taMN-DNA features can accurately distinguish early-stage HCC patients from HDs with high sensitivity. In both human and murine models, there is a significant relationship between these signatures and tumor presence, suggesting that taMN- DNA features might reflect the diseased state across mammalian species. Research sponsor: Timing Biotech. Citation Format: Hui Lin, Haobo Sun, Xingyun Yao, Xiaoxiao Fan, Fei Meng, Honghao Liang, Xiaofei Gao. Leveraging micronuclei DNA from erythrocytes for early detection of hepatocellular carcinoma [abstract]. In: Proceedings of the AACR Special Conference: Liquid Biopsy: From Discovery to Clinical Implementation; 2024 Nov 13-16; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(21_Suppl):Abstract nr B073.