DNA Isolation Techniques Research Articles

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.

Oxygen exposure decreases the yield of high-molecular-weight DNA from some anaerobic bacteria and bacterial communities during DNA extraction

ObjectivesThe central challenge in third-generation sequencing lies in meeting the requirements for DNA quality (integrity and purity) and quantity. Therefore, novel improvements in DNA extraction methods are needed to satisfy these requirements. We reasoned that in anaerobic microbial communities, the presence of certain strict anaerobes containing oxygen-activated DNase activity might contribute substantially to the poor integrity of extracted metagenomic DNA (or genomic DNA from some pure cultures) if exposed to air. MethodsTo test this hypothesis, we developed an enhanced genomic and metagenomic DNA isolation technique that we applied to a specifically chosen set of both strict and aerotolerant anaerobes, as well as to the hindgut microbiota of a herbivorous marine fish. ResultsConsidering the quality (or degradation) of extracted DNA obtained under anaerobic versus aerobic conditions, we found that DNA extracted aerobically from cells of some strict anaerobes showed more degradation of high molecular weight DNA than analogous preparations under anaerobic conditions. In contrast, with the selected aerotolerant anaerobes, no discernible difference was found between the molecular sizes of DNA extracted aerobically and anaerobically. Metagenomic DNA extracted from the fish hindgut microbiota showed higher yields and better quality under anaerobic conditions compared to aerobic conditions. ConclusionOur study effectively demonstrates the advantages of our improved extraction protocol in anaerobic conditions. This is evident through the improved quality of extracted DNA. Such findings may be valuable for studies, especially metagenomic studies, where the quality and quantity of DNA are crucial for downstream analysis.

Efficiency Evaluation of DNA Isolation Techniques in Fins of Channa marulius Using PCR Amplification and NanoDrop

Highly pure and good-quality extracted DNA is vital for PCR amplification, gene sequencing, and species identification. Seven genomic DNA isolation techniques were used to isolate the DNA from the fins of Channa marulius. The concentration and purity of isolated DNA were assessed with PCR amplification of the mitochondrial COI gene and NanoDrop. The phenol-chloroform method was found significantly (P<0.05) higher in concentration (1001 ng.ul-1) as compared to all other six evaluated DNA isolation methods. In terms of purity of isolated DNA GeneJET, the Genomic DNA Purification Kit was found significantly (P<0.05) higher among all analyzed methods. Both the Phenol chloroform method and GeneJET Genomic DNA Purification Kit were successfully used in PCR amplification. Concluding that the Phenol chloroform method and GeneJET Genomic DNA Purification Kit may be successfully used in PCR amplification. The phenol-chloroform method and GeneJET Genomic DNA Purification Kit proved the best in the extraction of DNA in high concentration with maximum yield and purity from fins of C. marulius and can be helpful to isolate DNA from fish species because a small quantity of fin tissues is required for DNA isolation without harm to fish. Moreover, data reported from this study about DNA isolation techniques may be used for molecular approaches like PCR amplification and gene sequencing of fishes.

Biological Diversity of Seawater Microalgae Isolated from Ujung Genteng Sukabumi and Their Novel Genomic DNA Isolation Technique

Biological Diversity of Seawater Microalgae Isolated from Ujung Genteng Sukabumi and Their Novel Genomic DNA Isolation Technique

Perbandingan Teknik Isolasi DNA pada Daun dan Kayu Sonokeling (Dalbergia latifolia)

Sonokeling (Dalbergia latifolia) is a hardwood plant, resistant to termite attack, its wood has beautiful patterned fibers and textures, so it is widely used for furniture in luxury categories with high economic value. According to CITES (Convention on International Trade in Endagered Species of Wild Fauna and Flora), rosewood trade status is included in appendix II. In the trade of rosewood in both domestic and international markets, there are relatively many obstacles and difficulties in identifying the origin of rosewood. It is very important to establish a reference on the identity of wood based on the location of origin as an effort to prevent illegal trade, namely by using a molecular-based identification approach (DNA fingerprinting). In the isolation of rosewood DNA, special techniques are required due to its hard wood characteristics. This study aims to compare DNA isolation techniques from rosewood and rosewood leaves using the CTAB (Cetyl Trimethyl Ammonium Bromide) protocol and the DNA Mini Kit Plant protocol. The research used molecular biology-based identification methods. PCR results showed that the DNA isolation protocol using the commercial kit outperformed the CTAB protocol for both leaf and wood samples. DNA isolation from wood samples was more difficult than leaf samples. Overall, the best protocol for DNA isolation from rosewood leaves and wood was to use a commercial kit.

Environmental DNA Isolation, Validation, and Preservation Methods.

Environmental DNA (eDNA) workflows contain many familiar molecular-lab techniques, but also employ several unique methodologies. When working with eDNA, it is essential to avoid contamination from the point of collection through preservation and select a meaningful negative control. As eDNA can be obtained from a variety of samples and habitats (e.g., soil, water, air, or tissue), protocols will vary depending on usage. Samples may require additional steps to dilute, block, or remove inhibitors or physically break up samples or filters. Thereafter, standard DNA isolation techniques (kit-based or phenol:chloroform:isoamyl [PCI]) are employed. Once DNA is extracted, it is typically quantified using a fluorometer. Yields vary greatly, but are important to know prior to amplification of the gene(s) of interest. Long-term storage of both the sampled material and the extracted DNA is encouraged, as it provides a backup for spilled/contaminated samples, lost data, reanalysis, and future studies using newer technology. Storage in a freezer is often ideal; however, some storage buffers (e.g., Longmires) require that filters or swabs are kept at room temperature to prevent precipitation of buffer-related solutes. These baseline methods for eDNA isolation, validation, and preservation are detailed in this protocol chapter. In addition, we outline a cost-effective, homebrew extraction protocol optimized to extract eDNA.

Analysis of microbial composition and sharing in low-biomass human milk samples: a comparison of DNA isolation and sequencing techniques

Human milk microbiome studies are currently hindered by low milk bacterial/human cell ratios and often rely on 16S rRNA gene sequencing, which limits downstream analyses. Here, we aimed to find a method to study milk bacteria and assess bacterial sharing between maternal and infant microbiota. We tested four DNA isolation methods, two bacterial enrichment methods and three sequencing methods on mock communities, milk samples and negative controls. Of the four DNA isolation kits, the DNeasy PowerSoil Pro (PS) and MagMAX Total Nucleic Acid Isolation (MX) kits provided consistent 16S rRNA gene sequencing results with low contamination. Neither enrichment method substantially decreased the human metagenomic sequencing read-depth. Long-read 16S-ITS-23S rRNA gene sequencing biased the mock community composition but provided consistent results for milk samples, with little contamination. In contrast to 16S rRNA gene sequencing, 16S-ITS-23S rRNA gene sequencing of milk, infant oral, infant faecal and maternal faecal DNA from 14 mother-infant pairs provided sufficient resolution to detect significantly more frequent sharing of bacteria between related pairs compared to unrelated pairs. In conclusion, PS or MX kit-DNA isolation followed by 16S rRNA gene sequencing reliably characterises human milk microbiota, and 16S-ITS-23S rRNA gene sequencing enables studies of bacterial transmission in low-biomass samples.

The Presence and Treatment of DNA isolation, PCR and Gel Electrophoresis Techniques in Biology Curricula, Textbooks and University Placement Exams: Are We Keeping up with the Pace of Biotechnology?

Understanding the basic applications of biotechnology is important in order to respond effectively to challenges related to GMOs, forensic science, bioinformatics, and the COVID-19 pandemic, which highlights the importance of biotechnology education. This study aims to examine the presence and treatment of biotechnological techniques (DNA isolation, PCR, and Gel Electrophoresis) in biology curricula, aligned biology textbooks, and university placement exams). The study adopts a qualitative case study approach, employing a document analysis method. Turkish National Biology Curriculum published in 2013 (BC-2013) and 2018 (BC-2018 and SBC-2018), curricula-aligned Biology Textbooks, and University Placement Biology Exam Questions (2016-2022) are used as documents. The results, regarding the examination of BC and SBC showed that the basic biotechnological methods were only included in the explanations of the SBC-2018. Regarding the textbooks, it was found out that 12nd grade textbooks include PCR and Gel Electrophoresis but not DNA isolation. Contents of these textbooks revealed some issues when examined in terms of textbook examination criteria. The biology questions in the university placement exams showed that they do not include biotechnological techniques or do not require knowledge of these techniques to choose the right option. As a result, this research highlights the challenges related to the current state of basic biotechnology techniques and emphasizes that curricula, aligned textbooks, and university entrance exams struggle to keep up with the pace of biotechnology. The study suggests updating biology curricula and aligned textbooks in terms biotechnological techniques and organizing them coherently to avoid scientific inaccuracies. It also suggests that the questions on university placement exams be revised to include more questions related to present-day biotechnology challenges and advancements

Analysis of Purity and Concentration of DNA Isolated in Dragonfly (Onychogomphus forcipatus)

Analysis of the purity and concentration of DNA isolation results was carried out using samples of dragonflies (Onychogomphus forcipatus) species of insects. This research is important because there are many kinds of research in the molecular field that require good DNA isolation techniques to support the success of the research. The purpose of this paper is to provide information about the quality of the isolated DNA seen using the purity and concentration parameters. The extraction method uses the centrifuge column method. The isolated data were analyzed statistically using the average test. The results of DNA isolation showed that the average concentration value was in the range of 60.10 – 69.95. The analysis of the purity of the isolated DNA read at the A260/A280 ratio was in the range of 1.817 – 1.929, while the purity analysis at the A260/A230 ratio was in the range of 0.760 – 0.822. Based on the results of the study, it can be concluded that the analysis of the purity and concentration of DNA from the isolation of dragonfly samples was in a good category at the A260/A280 ratio, while the purity read at the A260/A230 ratio was below the value categorized as good.

Comparison of seven genomic DNA isolation techniques from internal organs, gills and muscle tissues of Notopterus notopterus (Notopteridae) using PCR amplification and Nanodrop

The techniques and the principles used in DNA isolation play a vital role in the obtaining of a purified genetic material. In present study, we investigated the efficiency of seven genomic DNA isolation techniques in terms of isolated DNA concentration, yield and purity from internal organs, gills and muscle tissues of Notopterus notopterus. Isolated DNA quality was analysed through Nanodrod and PCR using mitochondrial COI genetic markers. Results showed that GeneJET Genomic DNA Purification Kit was found significantly higher in terms of isolated DNA concentration (1200-1288 ng.ul-1), yield (257.6 ng.ul-1) and purity (1.91-2.00), and also successful in PCR amplification as compared to other evaluated six traditional DNA isolation methods. The nucleotide minimum bands range was observed 200 base pairs in heart sample and maximum 600 base pair was observed in intestine. There is no data on description of parameters analysed in this work to date, neither the evaluation of isolated DNA using PCR amplification of mitochondrial COI gene for the species N. notopterus. Present study also revealed that the traditional DNA isolation methods are the secondary choice for isolation of DNA. The data of present study also indicated that the GeneJET Genomic DNA Purification Kit is useful for DNA isolation and can be used best in genetic applications for fishes.

Virtual Experiments of DNA Isolation and Hybridoma Techniques to Improve Students' Critical Thinking Ability

Virtual learning during the Covid-19 pandemic always requires innovation from educators to present effective and interesting learning. The case method is an alternative learning method that can be used to increase awareness responses to problems contextually. This study aims to determine the effectiveness of implementing the use of virtual DNA isolation experiments and hybridoma techniques on students' critical thinking skills. The research was conducted on students majoring in Integrated Science, Semarang State University in the 2021-2022 academic year. Samples from the population are divided into experimental class and control class. The research instrument used to measure critical thinking skills uses a test. The results of the assessment of critical thinking skills were analyzed using quantitative analysis. The results showed that the experimental class obtained an average critical thinking ability of 87.86 and the control class obtained an average critical thinking ability of 73.86. The results of the posttest difference test analysis between the experimental class and the control class obtained a sig. (2-tailed) 0.000 less than the t table significance value of 0.05. Based on these results it can be concluded that the implementation of the use of virtual DNA isolation experiments and hinridoma techniques is effective in increasing students' critical thinking skills. The novelty of the results is virtual experiments of DNA Isolation and Hibridoma can be an alternative to virtual practice for schools with incomplete laboratory facilities.

Analysis of Purity and Concentration of DNA Isolation Results on Chondroitin Samples

Analysis of purity and concentration of isolated DNA is a way to see the quality of isolated DNA. This research was conducted as an initial stage to give. Information about research conducted in future research. The purpose of this research is to provide references and information regarding DNA isolation techniques in chondroitin samples, so that they can be used in similar studies. The sample consisted of a sample of chondroitin which was weighed 50 mg and tested 10 times. The isolation method used in this study is the centrifuge column isolation method, while the purity and concentration analysis were analyzed using a nano photometer which was read at a wavelength of A260/A280. The value shown from the nano photometer was then analyzed statistically using the average test to determine the interpretation of the results from the data obtained. The results of DNA isolation obtained that the DNA concentration values ​​tested were in the range of 39.10 - 54.70 with an average concentration value of 45.15. The value of the purity of the isolated DNA tested was in the range of 2.16 – 2.28 with an average purity value of 2.22. Based on the results of the DNA confirmation test of the isolation carried out using real-time PCR, it showed that the isolated sample was amplified at a value of Ct 36.43 while the positive control was amplified at Ct 32.49. Based on the research results, it was found that all samples tested showed good average values ​​of DNA concentration and purity so that the results of the DNA isolation tested could be used as templates in real-time PCR analysis.

A rapid and efficient technique for the isolation of Bacillus genomic DNA using a cocktail of peptidoglycan hydrolases of different type.

The paper suggests a rapid and efficient technique for isolation of genomic DNA from the bacteria of the genus Bacillus, which is based on the hydrolysis of cell wall peptidoglycan by a cocktail of peptidoglycan hydrolases of different type (L,D-peptidase and N-acetylmuramidase). The comparing of conventional techniques for the isolation of genomic DNA using: a microwave treatment; a treatment with ionic detergents (SDS, CTAB) or a chaotropic agent (GuSCN); and enzymatic hydrolysis (nonspecific, with proteinase K, or specific, with peptidoglycan hydrolases) conducted on Bacillus megaterium, B. subtilis, B. licheniformis, B. cereus showed that the most effective ones were techniques based on the specific hydrolysis of cell wall peptidoglycan. The highest efficiency of hydrolysis was obtained with an enzyme cocktail consisted of hen egg muramidase (HEWL) and highly active phage-specific L,D-peptidase EndoRB49 revealed a pronounced synergism between the peptidase and the muramidase. The cocktail treatment of Bacillus cells could be reduced to 10min without affecting the yield of nucleic acids. The quality of DNA preparations was assessed using the restriction and PCR assays, as well as agarose gel electrophoresis. Using peptidoglycan hydrolases of different type, which have a good synergy, makes the technique very efficient and perspective for the application when rapid and effective disintegration of cell wall is crucial to avoid adverse effects of macromolecular denaturation.

P452 Estimation of the detection limit of extracted Candida DNA from spiked EDTA blood sample

Poster session 3, September 23, 2022, 12:30 PM - 1:30 PMBackgroundCandidemia caused by genus Candida, are opportunistic pathogens with life-threatening infection in immunocompromised individuals with a mortality rate being 40%–54% in patients. Blood culture is not a reliable and rapid method for the diagnosis of candidemia as its sensitivity is as low as 50%. Molecular diagnosis is an alternative to conventional culture for early detection of candidemia. An appropriate DNA isolation technique is needed to obtain high purity DNA from blood specimens and improves the sensitivity of the polymerase chain reaction (PCR). In this study, two different methods were used to extract Candida DNA from spiked EDTA blood specimens, the In-house phenol-chloroform and potassium acetate method.ObjectivesDNA extraction from the spiked blood samplesQuantification of the Candida DNA extracted from the spiked sampleComparison of the extracted DNA by phenol-chloroform and potassium acetate methodMethodsA total of 5 ml of EDTA blood samples from healthy volunteers were spiked with 104-108 Candida albicans cells (ATCC 90028) to determine the detection limit of our extraction method. DNA was extracted from whole blood using phenol-chloroform and the potassium acetate methods which involves pretreatment with erythrocytes, leukocytes and fungal lysis buffer. DNA from C. albicans were amplified using ITS1 and ITS4 based primers. PCR products were visualized by agarose gel electrophoresis.ResultsThe spiked Candida cells taken were from 104-108 yeast cells. The total extracted DNA by phenol chloroform extraction method ranged from 18.29 to 51.64 ng/μl respectively. Whereas the total extracted DNA from potassium acetate extraction method ranged from 15.58 to 32.30 ng/μl respectively. The presence of a 535 base pair (bp) product was considered positive (Figs. 1 and 2). The lowest detection limit of PCR has been determined as 106-108 Candida cells for our spiked samples.ConclusionIn our study, till date, the extracted quantity of DNA was found to be higher by phenol-chloroform method as compared to the potassium acetate method. Greater numbers and processing are required to obtain conclusive data.

Rapid and inexpensive method of PCR ready DNA isolation from human peripheral blood and saliva

BackgroundThe isolation of nucleic acids is a frequently performed procedure in the molecular biology area. Although several rapid DNA isolation techniques from human peripheral blood and saliva have been developed, there are still some disadvantages – volume, time, cost, and yield are a few notable ones. ObjectiveWe aim to develop a rapid and inexpensive method to isolate high-molecular-weight genomic DNA from human peripheral blood and saliva that can be used for molecular biology experiments. MethodsFive DNA isolation methods with slightly varying protocols were used. High-quality DNA obtained from one specific method was further amplified by PCR and the template with good amplification was further used for performing RFLP and sequencing. ResultsOut of 5 different isolation methods (R1 to R5), DNA obtained from the R4 was of good quality (molecular weight is > 10 kb and 260/280 ratio is 1.89 ± 0.2), which allows successful PCR amplification and good separation in Restriction Fragment Length Polymorphism analysis. Sequencing by the Sanger Sequencing produced a good readable sequence of an amplified fragment from Method R4 DNA. ConclusionIn the present study we have developed a simple, rapid, and cost-effective DNA isolation method, which uses low sample volume and yields good quantity and high-quality product. The DNA obtained is highly fit for molecular genetics research applications.

Evaluation and Comparison of Genomic DNA Extraction Methods and PCR Optimization on Archival Formalin-Fixed and Paraffin-Embedded Tissues of Oral Squamous Cell Carcinoma.

Recovery and amplification of nucleic acids from archived formalin-fixed tissue samples is the most developing field in retrospective genetic studies. We compared different deparaffinization methods and DNA isolation techniques, and intergroup comparisons were performed to evaluate the effectiveness of different storing methods for archival OSCC samples based on obtained mean DNA quantity, quality, and PCR amplification of the P53 gene. The study comprised 75 archival histologically diagnosed OSCC samples which were divided into Group I: Formalin-fixed paraffin-embedded tissue blocks and Group II: Long-term formalin-fixed tissue. A comparison of different deparaffinization methods showed that xylene deparaffinization is an efficient method to obtain suitable DNA. Comparing different DNA isolation techniques illustrated that the conventional phenol–chloroform method gives better integrity to DNA in contrast with the kit method. Comparison between FFPET and long-term FFT samples demonstrated that samples fixed in formalin overnight and embedded in wax yield better quality and quantity DNA in comparison with long-term samples fixed in formalin. To obtain suitable integrity of DNA, tissue samples should be stored by fixing in formalin overnight followed by preparation of paraffin tissue blocks, deparaffinization by xylene, and subjecting them to the conventional phenol–chloroform DNA isolation protocol.

DNA EXTRACTION TECHNIQUES FOR SOME WILD SPECIES FROM UZBEKISTAN

Although DNA extraction is already a routine procedure and protocols have been developed for a large number of plant species, various plant tissues and organs, solving the problem of extracting pure undegraded DNA from plant is the main step in molecular approaches for study plant organisms. The extraction of high-quality DNA from plant materials is an important factor for DNA barcoding. The effective DNA extraction protocol for samples with high content of admixture, which prevents the restriction reaction and the PCR, or degraded DNA should be established for each case. This work presents the comparison of DNA isolation techniques from silica gel dried and herbarium samples of some wild species collected in Uzbekistan. In this work, we optimized the technique for isolating the total DNA, on the examples of 16 representatives of Salvia, Dracocephalum, Phlomoides, Tulipa, Iris, Allium genera from Uzbekistan. The results of a comparative analysis of three methods of DNA extraction from leaf tissues of wild plants are represented in this article. DNA amount and purity degrees were measured using spectrophotometer. The efficiency of the DNA extraction method was assessed according to two criteria: the quantitative parameters -DNA concentration (ng/μl), and the qualitative parameters - DNA purity (nm). The DNA concentration variate from 758.1 ng/μl obtained with the CTAB method to 13.89 ng/μl obtained with commercial column extraction kits. λ = 260/280 ratio is range from 1.7 to 1.98 nm. Additionally, PCR amplification was realized to reflect the validation of the DNA isolation protocol, using specific primers of four chloroplast (rbcL, matK, psbA, trnL-F) and one nuclear (ITS) plant DNA barcode regions. The sequence length was 300–800 bp appropriately.

The untapped potential of macrofossils in ancient plant DNA research.

SummaryThe rapid development of ancient DNA analysis in the last decades has induced a paradigm shift in ecology and evolution. Driven by a combination of breakthroughs in DNA isolation techniques, high‐throughput sequencing, and bioinformatics, ancient genome‐scale data for a rapidly growing variety of taxa are now available, allowing researchers to directly observe demographic and evolutionary processes over time. However, the vast majority of paleogenomic studies still focus on human or animal remains. In this article, we make the case for a vast untapped resource of ancient plant material that is ideally suited for paleogenomic analyses: plant remains, such as needles, leaves, wood, seeds, or fruits, that are deposited in natural archives, such as lake sediments, permafrost, or even ice caves. Such plant remains are commonly found in large numbers and in stratigraphic sequence through time and have so far been used primarily to reconstruct past local species presences and abundances. However, they are also unique repositories of genetic information with the potential to revolutionize the fields of ecology and evolution by directly studying microevolutionary processes over time. Here, we give an overview of the current state‐of‐the‐art, address important challenges, and highlight new research avenues to inspire future research.

Optimisation of DNA isolation and PCR techniques for beetle (Order: Coleoptera) specimens

Optimisation of DNA isolation and PCR techniques for beetle (Order: Coleoptera) specimens

Rapid Detection of Transposon-Induced Genome Rearrangements.

This chapter details the techniques used to detect transposon-induced genome rearrangements. Here, we describe a rapid DNA isolation technique, PCR amplification, and a novel High Efficiency Agarose Gel Electrophoresis Method (HEA-GEM).