DNA Preparations Research Articles

Genetic diversity and breed-informative SNPs identification in domestic pig populations using coding SNPs.

Background: The use of breed-informative genetic markers, specifically coding Single Nucleotide Polymorphisms (SNPs), is crucial for breed traceability, authentication of meat and dairy products, and the preservation and improvement of pig breeds. By identifying breed informative markers, we aimed to gain insights into the genetic mechanisms that influence production traits, enabling informed decisions in animal management and promoting sustainable pig production to meet the growing demand for animal products. Methods: Our dataset consists of 300 coding SNPs genotyped from three Italian commercial pig populations: Landrace, Yorkshire, and Duroc. Firstly, we analyzed the genetic diversity among the populations. Then, we applied a discriminant analysis of principal components to identify the most informative SNPs for discriminating between these populations. Lastly, we conducted a functional enrichment analysis to identify the most enriched pathways related to the genetic variation observed in the pig populations. Results: The alpha diversity indexes revealed a high genetic diversity within the three breeds. The higher proportion of observed heterozygosity than expected revealed an excess of heterozygotes in the populations that was supported by negative values of the fixation index (FIS) and deviations from the Hardy-Weinberg equilibrium. The Euclidean distance, the pairwise FST, and the pairwise Nei's GST genetic distances revealed that Yorkshire and Landrace breeds are genetically the closest, with distance values of 2.242, 0.029, and 0.033, respectively. Conversely, Landrace and Duroc breeds showed the highest genetic divergence, with distance values of 2.815, 0.048, and 0.052, respectively. We identified 28 significant SNPs that are related to phenotypic traits and these SNPs were able to differentiate between the pig breeds with high accuracy. The Functional Enrichment Analysis of the informative SNPs highlighted biological functions related to DNA packaging, chromatin integrity, and the preparation of DNA into higher-order structures. Conclusion: Our study sheds light on the genetic underpinnings of phenotypic variation among three Italian pig breeds, offering potential insights into the mechanisms driving breed differentiation. By prioritizing breed-specific coding SNPs, our approach enables a more focused analysis of specific genomic regions relevant to the research question compared to analyzing the entire genome.

Quantitative detection of the Ralstonia solanacearum species complex in soil by qPCR combined with a recombinant internal control strain

ABSTRACT The detection of soil-borne pathogens by quantitative PCR (qPCR) has been challenging due to the pronounced influence of soil type on DNA extraction and PCR reactions. In the present study, we developed a novel qPCR system and an internal sample process control (ISPC) strain, RsPC, for the detection of Ralstonia solanacearum species complex (RSSC), the pathogens causing bacterial wilt. Specific primers and TaqMan probe were designed based on analyses of 16S rRNA gene sequences from 581 Ralstonia genomes, and the RsPC was constructed by insertion of an artificial fragment, which consisted of two fragments from the kanamycin-resistant gene and the gfp gene, into the chromosome of a phylogenetically closely related strain, Ralstonia pickettii JCM 5969. The qPCR target sequences of RSSC and RsPC shared primers; however, different TaqMan probes were used to distinguish them from each other. The interaction assay between ISPC and target DNA showed no influence on sensitivity when their difference in concentration was <10 4 -fold. We tested 10 different soils with co-spiked RsPC and R. pseudosolanacearum LMG 9673 and found comparable recovery efficiencies (REs) of two strains in most samples, and the REs of LMG 9673 after correction by RsPC were closer to theoretical values. The RE of LMG 9673 improved most (2.98-fold) in a heavy clay soil. Similar results were obtained in three representative soils co-spiked with RsPC and LMG 9673 (at three concentrations), R. solanacearum NCPPB 325, and R. syzygii LLRS-1, respectively . The qPCR system and ISPC strain developed in this study could be applied for the accurate detection of RSSC in soil, and similar ISPCs can be developed in the future for other soil-borne animal and plant pathogens. IMPORTANCE DNA-based detection and quantification of soil-borne pathogens, such as the Ralstonia solanacearum species complex (RSSC), plays a vital role in risk assessment, but meanwhile, precise quantification is difficult due to the poor purity and yield of the soil DNA retrieved. The internal sample process control (ISPC) strain RsPC we developed solved this problem and significantly improved the accuracy of quantification of RSSC in different soils. ISPC-based quantitative PCR detection is a method especially suitable for the quantitative detection of microbes in complex matrices (such as soil and sludge) containing various PCR inhibitors and for those not easy to lyse (like Gram-positive bacteria, fungi, and thick-wall cells like resting spores). In addition, the use of ISPC strains removes additional workload on the preparation of high-quality template DNA and facilitates the development of high-throughput quantitative detection techniques for soil microbes.

Molecular Findings on Plasma Cell-Free DNA Analysis Among Adults with Histiocytic Neoplasms

Introduction The discovery of MAPK/ERK pathway mutations has revolutionized the care for patients with histiocytic neoplasms. However, mutational analysis on biopsy specimens is often limited by low tumor cellularity leading to testing failure or disease involvement of difficult-to-reach sites (e.g., central nervous system). Recently, molecular analysis of cell-free DNA (cfDNA) has shown promise as a viable tool for discovering driver mutations in multiple cancers. A recent study of mainly BRAF-mutated non-Langerhans cell histiocytosis (LCH) showed 50% concordance between tumor and plasma cfDNA analysis for BRAF-V600E. However, it is unknown whether cfDNA has a role in the evaluation of cases that harbor mutations beyond BRAF-V600E. We conducted this study to address this unanswered question using a large cohort of individuals with diverse histiocytic neoplasms from a tertiary-care institution. Methods We included 40 cases with histiocytic neoplasms with stored plasma and germline DNA in this study. Tissue molecular analysis was conducted using a targeted next generation sequencing (NGS) using commercial assays or whole exome sequencing (WES) using an in-house assay. A custom panel of 226 genes, including exonic and intronic regions, was used for the cfDNA assay in this study. To avoid cfDNA false positives, germline mutations identified from normal DNA sequencing were filtered using a BostonGene ®-developed error reduction algorithm.The total length of the analyzed genomic regions was 1.56 MB. For samples with an input greater than 30 ng, a 0.2% variant allele frequency (VAF) limit of detection (LOD) was used; a 0.4% VAF LOD was used for samples less than 30 ng. Results The final analysis cohort included 38 patients with histiocytic neoplasms after exclusion of 2 cases (failure of germline DNA library preparation and mismatch of germline and somatic DNA, respectively). Disease types included Erdheim-Chester disease (ECD; n=13, 34%), Langerhans cell histiocytosis (LCH; n=13, 34%), and Rosai-Dorfman disease (RDD; n=12, 32%). 24 cases were multisystem (63%), and the remaining were single-system. Commonly involved organs included bone (n=25, 66%), kidney (n=12, 32%), skin (n=8, 21%), central nervous system (n=7, 18%), and pituitary (n=7, 18%). Most cases (n=31, 82%) underwent plasma collection pre-treatment, 5 cases underwent collection after histiocytosis-directed therapy, and 2 while on treatment. Tissue NGS/WES data were available for 28 (74%) cases; 5 cases (13%) had insufficient tissue cellularity, and 5 (13%) did not undergo any tissue sequencing. Among those who underwent successful tissue sequencing assays, the most common common driver alterations was BRAF V600E (n=5, 26%). Other alterations included those in MAP2K1 (n=4, 21%), BRAF-fusions (n=3, 10%; partners UBDT2, RNF11, and LMTK2, respectively), and single cases of KRAS, NRAS and BRAF indel, respectively. cfDNA analysis showed at least one variant in 23 cases (60%). Correlation between tissue and cfDNA for known driver mutations was poor and concordant findings were only seen in one case with ECD harboring BRAF V600E in tissue as well as plasma (VAF 0.5%). Among the 5 cases where tissue mutational analysis had failed, cfDNA showed mutations in MUTYH, PTCH2, NOTCH3 SDHD, and TMEM127, respectively (Figure 1). Among the 5 cases where tissue molecular assessment could not be undertaken due to various reasons, cfDNA showed alterations in 4 cases, encompassing NOTCH1, TET2, BRCA1, MSH6, PIK3CD, and FUS genes. Conclusions In a large cohort of adults with diverse histiocytic neoplasms, we found poor correlation between tumor and plasma cfDNA molecular findings. The reason for the lack of correlation may be related to low tumor shedding especially among non- BRAF V600E cases. cfDNA analysis may have a role to identify potential drivers of pathogenesis among cases that are unable to successfully undergo tissue molecular analysis. Further studies are underway to characterize other novel alterations that were discovered in the cfDNA analysis.

Novel Approaches to Possible Targeted Therapies and Prophylaxis of Uterine Fibroids.

Uterine leiomyomas are the most common benign tumors in women of childbearing age. They may lead to problems of conception or complications during the gestational period. The methods of treatment include surgical (myomectomy and hysterectomy, embolization of arteries) and therapeutic treatment (ulipristal acetate, leuprolide acetate, cetrorelix, goserelin, mifepristone). Both approaches are efficient but incompatible with pregnancy planning. Therefore, there is a call for medical practice to develop therapeutical means of preventing leiomyoma onset in patients planning on becoming pregnant. Based on the analysis of GWAS data on the search for mononucleotide polymorphisms associated with the risk of leiomyoma, in meta-transcriptomic and meta-methylomic studies, target proteins have been proposed. Prospective therapeutic treatments of leiomyoma may be based on chemical compounds, humanized recombinant antibodies, vaccines based on markers of the uterine leiomyoma cells that are absent in the adult organism, or DNA and RNA preparations. Three different nosological forms of the disease associated with driver mutations in the MED12, HMGA2, and FH genes should be considered when developing or prescribing drugs. For example, synthetic inhibitors and vaccines based on matrix metalloproteinases MMP11 and MMP16 are expected to be effective only for the prevention of the occurrence of MED12-dependent nodules.

Serratia marcescens enzyme SME-2 isolated from sputum in New Zealand.

The Serratia marcescens enzymes (SMEs) are chromosomally encoded Ambler Class A carbapenem-hydrolysing β-lactamases, which distinctively express resistance to carbapenems while remaining susceptible to extended-spectrum cephalosporins. Global reports of SMEs are infrequent. Here we describe the isolation of an SME-2-producing S. marcescens from the sputum of a patient who was hospitalized at Christchurch Hospital, New Zealand. An immunosuppressed asthmatic patient who presented with shortness of breath and hypoxia grew S. marcescens from a sputum culture. Antimicrobial susceptibilities were determined by Phoenix, with MICs of meropenem and imipenem determined by Liofilchem® MIC gradient strips and interpreted according to EUCAST breakpoints. Investigation for carbapenemase was performed using Carba NP, modified CIM (mCIM) and GeneXpert® Carba-R. WGS was performed using the Illumina DNA Prep library kit and sequenced using MiSeq. The isolate showed an unusual susceptibility profile, including high-level resistance to meropenem and imipenem, while remaining susceptible to extended-spectrum cephalosporins. The Carba NP and mCIM were positive and WGS demonstrated the presence of a blaSME-2 gene located on the chromosome within the SmarGI1-1 genomic island. In addition, a blaSRT-like class C β-lactamase, aac(6')-Ic aminoglycoside-modifying enzyme and various multidrug efflux mechanisms were found. Phylogenetic core-genome analysis indicated no matching genome with RefSeq database strains. S. marcescens is an opportunistic pathogen of concern, harbouring a variety of intrinsic resistance mechanisms, including the potential for stable AmpC hyperproduction. Globally, SME-type carbapenemases have been infrequently reported; however, isolates carrying this mechanism could have limited treated options, having implications for patient management. To the best of our knowledge this is the first report of SME in New Zealand.

Determination of <i>G</i>/<i>C</i> Polymorphism at chr20:37352001 Position in Human Blood DNA Preparations by GlaI- and Bst2UI-PCR Analyses Methods

Single nucleotide polymorphism (SNP) is a change of one nucleotide by another. This change often leads to an emergence (or disappearance) of a site recognized by a certain restriction endonuclease. As a result amplification of DNA fragment using primers surrounding SNP point (containing either N1 or N2 nucleotide) followed by hydrolysis of the amplicon with this restriction enzyme will be different for three possible variants in a diploid genome (genotypes N1/N1, N1/N2 and N2/N2). This method of restriction fragments length polymorphism (RFLP) is widely used in the genetic studies. Earlier we have developed GlaI- and FatI-PCR analyses methods which allowed to carry out real-time PCR and showed it applicability for SNP determination. In the current work a new way to determine the single nucleotide polymorphism G/C by the Bst2UI-PCR analysis is proposed. GlaI- and Bst2UI-PCR analyses have been used to determine the frequency of G/C polymorphism variants at the chr20:37352001 position (according to GRCH38.p14 genomic assembly) in the blood DNA samples of 161 donors. The study included: 1) the isolation of leukocyte DNA from blood cells; 2) GlaI- and Bst2UI-PCR analyses of the DNA fragment chr20:37351957-37352083, 3) determination of cytosine and guanine at the chr20:37352001 position in the analyzed DNA preparations, and 4) comparative analysis of the obtained results. It has been shown that 68 donors (42.2%) have a heterozygous set of G/C at the chr20:37352001 position, 89 donors (55.3%) are homozygous by G, and 4 donors (2.5%) are homozygous by C. Thus, taking into account that blood cells have a diploid set of chromosomes, G to C replacement occurs in 76 out of 322 analyzed cases (23.6%). At the same time, from the results obtained it follows that the cytosine residue complementary to G at the chr20:37352001 position exists in methylated form (5-methylcytosine) in most of the DNA molecules, both in homo- and heterozygotes. The proposed method of Bst2UI-PCR analyses extends the possibilites of SNP determination using real-time PCR.

Determination of G/C Polymorphism at chr20:37352001 Position in Human Blood DNA Preparations by Methods of GlaI- and Bst2UI-PCR Analyses

Determination of G/C Polymorphism at chr20:37352001 Position in Human Blood DNA Preparations by Methods of GlaI- and Bst2UI-PCR Analyses

Emetic and Diarrheal Toxins Detection and Typing of Bacillus cereus in Food and Human Milk in Italy

Abstract Background Bacillus cereus, responsible for 1.4%-12% of all food poisoning outbreaks worldwide, produces several virulence factors, including toxins which are mainly found in raw and processed food, as starch-based and dairy products. It can cause two types of gastrointestinal diseases: emetic form, with nausea and vomiting which appears as soon as after food consumption, and diarrheal form, with abdominal cramps, which occurs after 8-16 h. Moreover, B. cereus is the most common pathogen in pasteurized human milk, a precious aid in neonatal care. The aim of this study was to investigate the prevalence of B. cereus toxins kinds using WGS and to detect in silico sequence types (ST) in isolates from food and human milk. Methods During B. cereus routine surveillance in Lombardy (Italy), a total of 41 isolates were collected from food (n = 16) and donor breast milk (n = 25) between 2022-2023, and then analysed with WGS. Libraries were prepared using the Illumina DNA Prep Kit and WGS was performed on the Illumina MiSeq. Using the BTyper tool, for each sample emetic and diarrheal toxins were identified and ST was assigned. Results All 41 isolates had at least one diarrheal toxin, specifically the nonhemolytic enterotoxin was found in all the isolates, the cytotoxin K was detected in 37% of isolates, and the hemolysin BL in 32%. Instead, the emetic toxin was found in only 3 samples isolated from food. Using in silico MLST, different STs were found with a higher prevalence of ST26 and ST144. Conclusions Diarrheal toxins were detected in all samples underling that B. cereus food infections play an important role in the public health and in the food industry. These aspects are even more significant when the pathogen is isolated from human breast milk, as it can induce severe infections in newborns. Key messages • The presence of B. cereus toxins in food and human milk underlines the importance of its ongoing monitoring and analysis. • Human breast milk has a high risk of B. cereus contamination which is the most frequent cause for its discarding.

A Simplified, Efficient and Rapid DNA Extraction Protocol from Rice Grains and Leaf

DNA extraction is very complicated with different plant species because the presence of secondary metabolites that hinder with DNA isolation processes and application like DNA restriction, Gene amplification, as well as gene cloning. A simple method for preparation of rice genomic DNA was developed. The current study expressed comparatively rapid, cost-effective, less time consuming, methods for DNA extraction from seed, young leaves and old leaves of rice genotypes without using liquid nitrogen, phenol, and β–mercaptoethanol in extraction buffer. Using this method, high quality and quantity of genomic DNA was get from 0.5 g of rice seed, young leaves and 75 days old leavesand extracted total nucleic acids (genomic DNA) were amplified by employing single sequence repeats (SSR) or microsatellite markers, which produced reproducible results.This protocol resolves the problems of DNA degradation, contamination, and low yield due to binding and/or coprecipitation with starches and polysaccharides.

Method to generate Holliday junction recombination intermediates via RecA-mediated four-strand exchange

DNA molecules that contain single Holliday junctions have served as model substrates to investigate the pathway in which homologous recombination intermediates are processed. However, the preparation of DNA containing Holliday junctions in high yield remains a challenge. In this work, we used a nicking endonuclease to generate gapped DNA, from which α-structured DNA or figure-8 DNA were created via RecA-mediated reactions. The resulting DNA molecules were found to serve as good substrates for Holliday junction resolvases. The simplified method negates the requirement for radioactive labelling of DNA, making the generation of Holliday junction DNA more accessible to non-experts.

ЕФЕКТИВНІСТЬ РІЗНИХ МЕТОДІВ ЕКСТРАКЦІЇ ДНК, ПРИДАТНОЇ ДЛЯ ПЛР ІЗ ГЕРБАРНИХ ЗРАЗКІВ ВОДОРОСТЕЙ РОДУ CLADOPHORA KÜTZ.

DNA extraction from algae presents unique challenges due to the production of various polysaccharides and polyphenols, which can act as PCR inhibitors. Consequently, optimizing DNA extraction methods becomes necessary for different groups of algae. The Cladophora genus, known for its high morphological variability influenced by environmental conditions and developmental stages, can benefit from molecular genetic analysis in addressing taxonomic and phylogenetic inquiries within the green algae family.
 In this context, the primary objective of this study was to select an appropriate DNA extraction method for herbarium specimens of Cladophora algae and evaluate the extracted DNA's suitability for PCR. The study compared two DNA extraction methods, namely the SureFood PREP Basic kit from R-Biopharm and the Quick DNA Mini Prep kit from Zymo Research. Subsequently, the isolated DNA was subjected to PCR using the universal primer iPBS 2080 with different reaction mixtures containing Dream-Taq polymerase and Sso7d DNA polymerase.
 The results revealed that the DNA concentrations and purification rates obtained from samples extracted using the SureFood PREP Basic kit were notably superior. However, PCR using these DNA samples did not yield positive results, likely due to the presence of inhibitors. On the other hand, DNA extracted using the Quick DNA Mini Prep kit exhibited lower purification rates but likely contained fewer DNA polymerase inhibitors. This allowed for successful amplification with the iPBS 2080 primer, utilizing the high-efficiency Sso7d polymerase, known for its resistance to inhibitors.

First Report of Fire Blight on Chinese hawthorn (Crataegus pinnatifida Bunge) Caused by Erwinia amylovora in Korea.

Fire blight is one of the destructive plant diseases caused by Erwinia amylovora and causes enormous economic losses worldwide. Fire blight was initially reported in apples, pears, and Chinese quince (Park et al. 2016; Myung et al. 2016a, 2016b) in Korea, but recent studies have reported new hosts such as apricot (Lee et al. 2021) and mountain ash (Lim et al, 2023). These reports indicate that fire blight is likely to disperse to new hosts in Korea. During the nationwide survey in June 2021, we observed typical symptoms of blossom blight and shoot blight on a Chinese hawthorn (Crataegus pinnatifida Bunge) just near an orchard (37°09'21.7"N, 127°35'02.6"E) in Icheon, Gyeonggi Province, where fire blight of Asian pear occurred. For identifying its causal agent, bacterial isolates were recovered after incubating at 28 ℃ for 24 hours on tryptic soy agar (TSA) medium (BD Difco, USA) from blighted leaves and shoots that were surface sterilized with 70% alcohol for 30 sec and homogenized in 500 µl of 10mM MgCl2. Pure cultures of white to mucoid colonies were grown on mannitol glutamate yeast extract (MGY) medium, a semi-selective medium for E. amylovora (Shrestha et al, 2003). Two isolates produced 1.5 kb amplicon through colony PCR using amsB primers (Bereswill et al. 1995). Two strains (CPFB26 and CPFB27) from the Chinese hawthorn produced amplicons identical to that from the TS3128 strain of E. amylovora, isolated from the pear tree and identified in 2016 (Park et al. 2016). For the partial 16s rRNA sequences, the total DNA of these two strains was extracted using the Wizard DNA prep kit (Promega, USA), and PCR was performed using fD1 (5'-AGAGTTTGATCCTGGCTCAG-3') and Rp2 (5'-ACGGCTACCTTGTTACGACTT-3') primer sets and further sequenced (Weisburg et al. 1991). These sequences belonged to the E. amylovora clade and were identified as E. amylovora in phylogenetic analysis (GenBank accession no. OP753569 and OP753570). Based on BLASTN analysis, CPFB26 and CPFB27 showed 99.78% similarity to the sequences of the E. amylovora strains TS3128, CFBP 1430, and ATCC 49946. To confirm pathogenicity of the isolates, 10 ㎕ bacterial suspensions (1.5 ⅹ 108 CFU/ml) was injected through the veins of the upper 2nd leaf of 3-month-old clone of apple rootstock (Malus domestica cv. M29) and incubated for six days at 28 ℃ in a chamber with 12 hours of light per day. Petioles and stems turned red hue, and the shoots finally blighted. To complete Koch's postulates, colonies were recovered on TSA medium from the inoculated apple rootstocks and verified through colony PCR for the amsB and A/B primer set (Powney et al. 2011). Hawthorn has been reported as an epidemiologically important alternate host plant of fire blight (van der Zwet et al. 2012). This study is the first to report fire blight caused by E. amylovora in Chinese hawthorn in Korea. Because Chinese hawthorn is natively distributed in Korea and is widely used as a landscaping tree (Jang et al. 2006), the findings of this study suggest that early monitoring could prevent the spread of fire blight through natural hosts.

Recombinant Production of Food Allergens in Yeast Pichia pastoris.

The methylotrophic yeast Pichia pastoris (P. pastoris) is one of the expression systems widely used to produce recombinant heterologous proteins. In this chapter, the methodology to produce recombinant food allergens in P. pastoris is described. The methodology begins with the preparation of competent P. pastoris cells followed by the transformation of the competent cells by electroporation as well as the preparation of plasmid DNA for transformation. Moreover, the screening of yeast transformants by direct PCR to ensure integration of allergen DNA followed by small-scale expression of recombinant allergen in yeast cells is also described.

Tracking the geographical origin of Plasmodium falciparum causing a rare severe case of malaria imported into Palestine, a zero-indigenous case area

BackgroundMalaria cases in non-endemic zero-indigenous case areas are most likely to have been imported whatever of the route of importation. In countries recently declared malaria-free and now without local transmission, imported cases remain a threat to re-introduction of the disease and a burden on the health system.Case presentationThree days after returning from a long trip to malaria- endemic countries; Abyei-Sudan, Chad and Uganda, a 41-year-old male resident from Jericho, Palestine, suffered paroxysms of fever, general fatigue, myalgia, arthralgia, headache, and a strong desire to vomit. Thin and thick Giemsa-stained blood smears were prepared and examined microscopically using oil immersion. Immature trophozoites (ring forms) were seen to parasitize approximately 10% of the erythrocytes revealing hyperparasitemia equivalent to > 100,000 parasites/ µl indicating severe malaria [1, 2]. The double chromatin configuration (headphones) and accolé (applique) position are both indicative of Plasmodium falciparum infection. The 18S rRNA- PCR targeting the rPLU6-rPLU5 region was used to confirm the diagnosis. The next-generation sequencing (NGS) method was carried out according to the manufacturer’s instructions (Illumina® DNA Prep, (M) Tagmentation kit (20060060), Illumina) to identify Plasmodium spp. Furthermore, NGS produced a whole-genome sequence of 22.8Mbp of the 14 chromosomes and 25Kbp of the apicoplast. A BLAST search of the apicoplast DNA and selected chromosomal DNA revealed that P. falciparum was the causative agent. The merozoite surface protein-1 (msp-1) was used to construct a phylogenetic tree of 26 P. falciparum, including the one isolated from the patient from Jericho, which clustered with the Sudanese isolate indicating genetic relatedness between the two.ConclusionThe travel history together with signs and symptoms of malaria, followed by prompt diagnosis using conventional microscopic inspection of Giemsa-stained films together with molecular DNA tracking tools like msp-1 were key means in tracking the place of origin of infection in the case of travel to multiple destination.

Ultra-short cell-free DNA fragments enhance cancer early detection in a multi-analyte blood test combining mutation, protein and fragmentomics.

Cancer morbidity and mortality can be reduced if the cancer is detected early. Cell-free DNA (cfDNA) fragmentomics emerged as a novel epigenetic biomarker for early cancer detection, however, it is still at its infancy and requires technical improvement. We sought to apply a single-strand DNA sequencing technology, for measuring genetic and fragmentomic features of cfDNA and evaluate the performance in detecting multiple cancers. Blood samples of 364 patients from six cancer types (colorectal, esophageal, gastric, liver, lung, and ovarian cancers) and 675 healthy individuals were included in this study. Circulating tumor DNA mutations, cfDNA fragmentomic features and a set of protein biomarkers were assayed. Sensitivity and specificity were reported by cancer types and stages. Circular Ligation Amplification and sequencing (CLAmp-seq), a single-strand DNA sequencing technology, yielded a population of ultra-short fragments (<100 bp) than double-strand DNA preparation protocols and reveals a more significant size difference between cancer and healthy cfDNA fragments (25.84 bp vs. 16.05 bp). Analysis of the subnucleosomal peaks in ultra-short cfDNA fragments indicates that these peaks are regulatory element "footprints" and correlates with gene expression and cancer stages. At 98 % specificity, a prediction model using ctDNA mutations alone showed an overall sensitivity of 46 %; sensitivity reaches 60 % when protein is added, sensitivity further increases to 66 % when fragmentomics is also integrated. More improvements observed for samples representing earlier cancer stages than later ones. These results suggest synergistic properties of protein, genetic and fragmentomics features in the identification of early-stage cancers.

Selection and optimization of the method for DNA isolation and purification from Corylus species for PCR analysis

Aim.The use of molecular genetic analysis tools requires DNA preparations of high purity. However, in the case of hazelnut plants (Corylus spp.), obtaining quality DNA is complicated by the presence of a significant amount of secondary metabolites in the leaf tissue. The aim of the study was to select and optimize the method for isolation and purification of hazelnut DNA for further use in PCR analysis. Methods. Three methods of DNA isolation using cetavlon as a detergent were used, which differ in the DNA purification steps: the commonly used protocol by Doyle, Doyle, 1987; a modified method with purification using silicon dioxide microparticles; and a method using the extraction of soluble metabolites, which precedes the DNA isolation step. Results. The first two methods were shown to be unsuitable for DNA isolation from dry hazelnut leaves due to a high amount of impurities. The last method allowed to remove a significant part of water-soluble organic substances at the first stage and obtain high-quality DNA preparations. Conclusions. The method for DNA isolation from Corylus spp. plants and hybrids was selected and optimized, which allows isolation of a sufficient amount of high-quality DNA from dried leaf tissue, suitable for further molecular genetic analysis using PCR.

АПРОБАЦИЯ НЕПРЯМОГО МЕТОДА ВЫДЕЛЕНИЯ ДНК ИЗ ПОЧВЫ ДЛЯ ИДЕНТИФИКАЦИИ БАКТЕРИАЛЬНЫХ СООБЩЕСТВ ЧЕРНОЗЕМОВ ВЫЩЕЛОЧЕННЫХ

The article presents data on the testing of an indirect method of DNA extraction from soil to identify the soil microbiota of leached chernozems based on the analysis of the 16s rRNA gene. For this method, the optimal amount of soil sample was set at 0.3 - 0.4 g. Isolation of DNA from samples depending on the depth of the layer showed that the highest concentration was obtained from samples from depths of 10-20, 20-30 cm. As a result of the analysis, preparations of total DNA were obtained in various concentrations and different optical purity.

Recognition of an Unnatural Base Pair by Tool Enzymes from Bacteriophages and Its Application in the Enzymatic Preparation of DNA with an Expanded Genetic Alphabet.

Unnatural base pairs (UBPs) have been developed to expand the genetic alphabet in vitro and in vivo. UBP dNaM-dTPT3 and its analogues have been successfully used to construct the first set of semi-synthetic organisms, which suggested the great potential of UBPs to be used for producing novel synthetic biological parts. Two prerequisites for doing so are the facile manipulation of DNA containing UBPs with common tool enzymes, including DNA polymerases and ligases, and the easy availability of UBP-containing DNA strands. Besides, for the application of UBPs in phage synthetic biology, the recognition of UBPs by phage enzymes is essential. Here, we first explore the recognition of dNaM-dTPT3 by a family B DNA polymerase from bacteriophage, T4 DNA polymerase D219A. Results from primer extension, steady-state kinetics, and gap-filling experiments suggest that T4 DNA polymerase D219A can efficiently and faithfully replicate dNaM-dTPT3, and efficiently fill a gap by inserting dTPT3TP or its analogues opposite dNaM. We then systematically explore the recognition of dNaM-dTPT3 and its analogues by different DNA ligases from bacteriophages and find that these DNA ligases are generally able to efficiently ligate the DNA nick next to dNaM-dTPT3 or its analogues, albeit with slightly different efficiencies. These results suggest more enzymatic tools for the manipulation of dNaM-dTPT3 and indicate the potential use of dNaM-dTPT3 for expanding the genetic alphabet in bacteriophages. Based on these results, we next develop and comprehensively optimize an upgraded method for enzymatic preparation of unnatural nucleobase (UB)-containing DNA oligonucleotides with good simplicity and universality.

A modified CTAB method for high-molecular-weight DNA preparation from deep-sea holothurians

ABSTRACT With the features as global distribution and all depths dwelling, holothurians function an ideal model for the study of deep-sea adaptation. Acquisition of high-molecular-weight DNA is the basic requirement for molecular biology analyses. However, extraction of high-molecular-weight DNA from deep-sea holothurians is difficult by degradation and contamination. Here, we presented a modified CTAB (lysis buffer with cetyl trimethyl ammonium bromide) method and compared its extraction efficiency for deep-sea holothurians with other five common methods, i.e. the Tiangen Biotech kit, Qiagen kit, Omega Bio-tek kit, Sangon Biotech kit, and SDS (lysis buffer with sodium dodecyl sulfate). Based on the results of yield, integrity, and purity, we found that the modified CTAB method is more suitable to extract high-quality DNA for deep-sea holothurians, with a large amount of yield, minimized degradation, and higher purity. This study provides a high-quality DNA of deep-sea holothurians for genome sequencing and further studies.

A Field Deployable Real-Time Loop-Mediated Isothermal Amplification Targeting Five Copy nrdB Gene for the Detection of 'Candidatus Liberibacter asiaticus' in Citrus.

Huanglongbing (HLB) is one of the most destructive diseases in citrus, which imperils the sustainability of citriculture worldwide. The presumed causal agent of HLB, 'Candidatus Liberibacter asiaticus' (CLas) is a non-culturable phloem-limited α-proteobacterium transmitted by Asian citrus psyllids (ACP, Diaphorina citri Kuwayama). A widely adopted method for HLB diagnosis is based on quantitative real-time polymerase chain reaction (qPCR). Although HLB diagnostic qPCR provides high sensitivity and good reproducibility, it is limited by time-consuming DNA preparation from plant tissue or ACP and the requirement of proper lab instruments including a thermal cycler to conduct qPCR. In an attempt to develop a quick assay that can be deployed in the field for CLas detection, we developed a real-time loop-mediated isothermal amplification (rt-LAMP) assay by targeting the CLas five copy nrdB gene. The rt-LAMP assay using various plant sample types and psyllids successfully detected the nrdB target as low as ~2.6 Log10 copies. Although the rt-LAMP assay was less sensitive than laboratory-based qPCR (detection limit ~10 copies), the data obtained with citrus leaf and bark and ACP showed that the rt-LAMP assay has &gt;96% CLas detection rate, compared to that of laboratory-based qPCR. However, the CLas detection rate in fibrous roots was significantly decreased compared to qPCR due to low CLas titer in some root DNA sample. We also demonstrated that the rt-LAMP assay can be used with a crude leaf DNA extract which is fully deployable in the field for quick and reliable HLB screening.