Method For Extraction Of Genomic DNA Research Articles

Comparison of Three Genomic DNA Extraction Methods from Sugarcane for Detection of Sugarcane White Leaf Phytoplasma

Comparison of Three Genomic DNA Extraction Methods from Sugarcane for Detection of Sugarcane White Leaf Phytoplasma

Abstract 7248: Simultaneous isolation and parallel analysis of genomic DNA and RNA for gene therapy

Abstract Introduction Viral vectors, such as adeno-associated vector (AAV), play a crucial role in delivering transgenes for gene therapy research. The success of vector transduction is vital for efficacy, which is measured through vector copy number (VCN) and transgene expression. In preclinical studies, both VCN and transgene expression are commonly tested from the same sample to ensure accuracy. We have reported a method for simultaneous high-quality genomic DNA and RNA extraction without splitting the sample, utilizing SPRI bead-based technology that has consistent performance across diverse tissue types and brain regions. Methods AAV was administered to mouse and non-human primate (NHP) models. At the conclusion of each experiment, tissue samples from the adrenal gland, brain, gonads, heart, kidney, liver, lung, skeletal muscle, spinal cord, and spleen were harvested. All tissue samples that demonstrated around 15 mm3 were snap-frozen in liquid nitrogen before nucleic acid extraction. The extraction process was carried out manually or on an automated liquid handler. The quality and quantity of DNA and RNA were then assessed from all tested tissues before downstream assay. Results Our study found that all mouse tissue types (n=72/tissue type) and NHP (n=11/tissue type) had an average gDNA and RNA yield that exceeded the minimum yield cutoff of 25 ng/μL for DNA and 6.25 ng/μL for RNA. We also analyzed DNA from tissues treated with two different AAV serotype-treated tissues in the mouse model, which showed that transduction across all the tissues was highly efficient for both AAV serotypes. Furthermore, we observed variations in transgene expression levels (copies/1 μg RNA) among different tissue types. Similar patterns of VCN and transgene expression were also observed in NHP models. Conclusions Efficient gene therapy depends on a safe and effective viral vector. The simultaneous extraction of gDNA and RNA simplifies the process, saves time, ensures quality, and facilitates integration into existing lab automation. This leads to consistent and error-resistant performance in analyzing vector copy and transgene levels. Citation Format: Han Wei. Simultaneous isolation and parallel analysis of genomic DNA and RNA for gene therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7248.

High-quality genomic DNA extraction methods of Yellow Spathoglottis Blume complex for next-generation sequencing

Abstract. Jasim JHM, Othman AS, Nordin FA, Talkah NSM. 2024. High-quality genomic DNA extraction methods of Yellow Spathoglottis Blume complex for next-generation sequencing. Biodiversitas 25: 654-663. Advancements in genomic research have spurred a growing interest in extracting high-quality genomic DNA, particularly from intricate plant species like the Yellow Spathoglottis Blume Complex. The preparation of genomic DNA high-quality from a plant sample is a crucial step for genomic and genetic analysis studies. A variety of genomic DNA extraction methods such as the traditional Hexadecyltrimethylammonium Bromide (CTAB) method and commercially available kits have been reported. Even so, they are either low purity and yield or costly. We develop a good-quality genomic DNA extraction method from fresh and dried leaves of the Yellow Spathoglottis Blume complex in Peninsular Malaysia, specifically tailored for Next-Generation Sequencing (NGS) applications. Three DNA extraction methods were compared traditional CTAB, modified CTAB, and the DNeasy Qiagen plant mini kit methods. The yield and quality of extracted DNA were assessed by PCR amplification using nuclear ETS and ITS genes performed to evaluate the success of DNA extraction. Additionally, the suitability of the modified CTAB method for NGS library preparation and sequencing was tested. It demonstrated better PCR amplification results, yielding high-concentration bands for both ETS and ITS primers. Furthermore, the modified CTAB method proved suitable for high molecular weight DNA extraction, without contamination. The extracted DNA from Spathoglottis aurea exhibited high molecular weight and passed the quality control assessment for NGS library preparation. Sequencing results demonstrated an average quality score of 36.0, with an accurate base call rate of approximately 99.9%. The developed method enables reliable genomic analysis and sequencing of the Yellow Spathoglottis Blume complex, contributing to further research and understanding of this species group.

An optimal genomic DNA extraction method for shoots of four Dendrocalamus species based on membership function analysis.

High-quality genomic DNA extraction is fundamental for the study of gene cloning and expression in plants. Therefore, this study evaluated several methods for extracting genomic DNA from shoots of four Dendrocalamus species to determine the optimal technique. Genomic DNA was extracted using three different methods: a commercial DNA extraction kit method, a modified cetyltrimethylammonium bromide method and a sodium dodecyl sulfate method. A membership function analysis was employed to compare these methods. The results demonstrated that the commercial DNA extraction kit method was the most effective and comprehensive approach for extracting genomic DNA from shoots of four Dendrocalamus species. Furthermore, this study provided valuable insights into optimizing techniques for extracting genomic DNA in other bamboo species.

Species identification method by a new non-invasive technique in Korean endangered terrestrial snail, Koreanohadra Koreana (Gastropoda: Mollusca)

Koreanohadra koreana (K. koreana) is an endemic species in South Korea that is listed as endangered. While the ecology and phylogenetics of K. koreana have been studied, its morphological similarity to the related species Koreanohadra kurodana (K. kurodana), can make species identification difficult. Furthermore, this has led to confusion when determining essential habitat information for the conservation of K. koreana. To bypass this issue, we have developed a non-invasive species identification method that can genetically differentiate between them. While there are already various non-invasive genomic DNA (gDNA) extraction methods that utilize the mucus from mollusks, they are limited as they require the target species to be physically located. To address this, in this investigation a method of extracting gDNA from the feces of snails was developed. The method utilized a primer set to amplify a cytochrome b fragment from K. koreana but not K. kurodana or other terrestrial snails. The feces of terrestrial snails could thus be used to obtain gDNA to a genetically usable level if collected within 5 days of excretion. This non-invasive species identification method using feces will help to facilitate genetic research without harming the endangered species and if the target species is not physically in the habitat. Moreover, K. koreana and K. kurodana could perhaps be further distinguished, using their habitat information to help facilitate essential conservation measures.

Comparative Study of Genomic DNA Extraction Methods for Common bean (Phaseolus vulgaris L.)

Common bean (Phaseolus vulgaris L.) is one of the significant grains used in the human diet, accounting for half of all grain legumes consumed globally. To enhance production, conventional breeding and molecular approaches have been used so far. An efficient and rapid genomic DNA extraction method is required for these molecular approaches. The aim of this study was to compare and optimize an efficient and rapid DNA extraction protocol for common bean. Modified cetyl trimethylammonium bromide (CTAB) and potassium chloride (KCL) extraction methods were used. The mean DNA yield per nanoliter was 209 µg from modified CTAB and 150.3 µg from the KCL method. The concentration of gDNA was significantly (P< 0.05) higher for the KCL method, which was 5.01 µg/µl and 2.09 µg/µl for the CTAB method. The obtained DNA was also pure, with an absorbance ratio at 260 nm to an absorbance of 280 nm (A260/280) of 1.75-2.23 for the KCL method and 1.86-2.09 for the modified CTAB method. Gel electrophoresis separation was used to evaluate the quality of the total DNA extracted by the present protocols. The results showed that intense bands close to the gel wells were obtained from both extraction methods. DNA isolated with the two methods was successfully used for PCR-based downstream analysis, which includes random amplified polymorphic DNA (RAPD) and simple sequence repeat (SSR) markers. In this study, it took approximately 150 minutes for KCL and 240 minutes for the CTAB for whole process. In contrast to the CTAB method, the KCL method uses inexpensive and less hazardous reagents and requires only ordinary laboratory equipment. Therefore, it is more convenient and economical than the traditional technique.

Portuguese Neonatal Screening Programme: A Retrospective Cohort Study of 18 Years of MS/MS.

The Portuguese Neonatal Screening Programme (PNSP) identifies patients with rare diseases through nationwide screening. Currently, 27 diseases are diagnosed, amongst which are 24 Inborn Errors of Metabolism (IEM), covering approximately 100% of neonates (1). In 2004, the national laboratory implemented a new screening method, tandem mass spectrometry (MS/MS) to test for amino acids and acylcarnitines. This new protocol revolutionized the PNSP and allowed for the analysis of an increased number of IEM, with clear improvements in treatment timings and clinical outcomes (2). From 2004 to 2022, 1 764 830 neonates were screened with MS/MS technology. Those who displayed biochemical profiles indicating an IEM were subjected to molecular characterization via genomic DNA extraction, PCR amplification, and direct Sanger sequencing method of dried blood spot samples. A cohort of 681 newborns were diagnosed with an IEM. MCAD deficiency is the most frequent, with 233 confirmed diagnoses, showing predominantly c.985A>G (p.K329E) mutation of the ACADM gene in homozygosity. Approximately 1/3 of the 33 confirmed cases of Glutaric Aciduria type I present homozygous for the c.1204C>T (p.Arg402Trp) mutation in GCDH. Around 60% of cases of MAT II/III deficiency display the dominant mutation of the MAT1A gene, c.791G>A (p.Arg264His). These genetic profiles and others were determined as diagnostic confirmation for 24 of the IEM screened. This data shows the molecular epidemiology of patients with confirmed IEM diagnosis identified by neonatal screening. Some diseases out of the scope of the PNSP were also detected as a differential diagnosis after biochemical suspicion in the dried blood spot sample. The retrospective analysis of the PNSP allows for an overview of 18 years of achievements accomplished by the national screening for IEM since MS/MS was implemented. For some pathologies with low incidence, it's difficult to trace a discernible pattern. However, presenting de novo mutations for these diseases might provide insights on how to approach different phenotypes. The aim of this work is to establish the molecular epidemiology of metabolic diseases screened.

Development of a rapid detection method for Karenia mikimotoi by using CRISPR-Cas12a.

Harmful algal blooms (HABs), mainly formed by dinoflagellates, have detrimental effects on marine ecosystems and public health. Therefore, detecting HABs is crucial for early warning and prevention of HABs as well as the mitigation of their adverse effects. Although various methods, such as light microscopy, electron microscopy, real-time PCR, and microarrays, have already been established for the detection of HABs, they are still cumbersome to be exploited in the field. Therefore, rapid nucleic detection methods such as recombinase polymerase amplification (RPA) and loop-mediated isothermal amplification (LAMP)-lateral flow dipstick (LFD) have been developed for monitoring bloom-forming algae. However, the CRISPR/Cas-based detection of HABs has yet to be applied to this field. In this study, we developed a method for detecting Karenia mikimotoi (K. mikimotoi), a typical ichthyotoxic dinoflagellate responsible for global blooms. Our method utilized Cas12a from Lachnospiraceae bacterium ND2006 (LbCas12a) to target and cleave the internal transcribed spacer (ITS) of K. mikimotoi, guided by RNA. We leveraged the target-activated non-specific single-stranded deoxyribonuclease cleavage activity of LbCas12a to generate signals that can be detected using fluorescence-read machines or LFDs. By combining RPA and LbCas12a with reporters, we significantly enhanced the sensitivity, enabling the detection of ITS-harboring plasmids at concentrations as low as 9.8 aM and genomic DNA of K. mikimotoi at levels as low as 3.6 × 10-5 ng/μl. Moreover, we simplified the genomic DNA extraction method using cellulose filter paper (CFP) by directly eluting the DNA into RPA reactions, reducing the extraction time to < 30 s. The entire process, from genomic DNA extraction to result reporting, takes less than an hour, enabling the identification of nearly a single cell. In conclusion, our method provided an easy, specific, and sensitive approach for detecting K. mikimotoi, offering the potential for efficient monitoring and management of K. mikimotoi blooms.

Insight into Increased Recovery and Simplification of Genomic DNA Extraction Methods from Dried Blood Spots.

There is no consensus on how to perform the manual extraction of nucleic acids from dried blood spots (DBSs). Current methods typically involve agitation of the DBSs in a solution for varying amounts of time with or without heat, and then purification of the eluted nucleic acids with a purification protocol. We explored several characteristics of genomic DNA (gDNA) DBS extraction such as extraction efficiency, the role of red blood cells (RBCs) in extraction and critical kinetic factors to understand if these protocols can be simplified while maintaining sufficient gDNA recovery. We found that agitation in a RBC lysis buffer before performing a DBS gDNA extraction protocol increases yield 1.5 to 5-fold, depending upon the anticoagulant used. The use of an alkaline lysing agent along with either heat or agitation was sufficient to elute quantitative polymerase chain reaction (qPCR) amplifiable gDNA in 5 minutes. This work adds insight into the extraction of gDNA from DBSs with the intention of informing a simple, standardized manual protocol for extraction.

An Effective and Low-Cost Method for Genomic DNA Extraction from Cyclanthera Pedata Species (A Nutraceutical Plant) without Liquid Nitrogen for Inter Simple Sequence Repeat Analyses

An Effective and Low-Cost Method for Genomic DNA Extraction from Cyclanthera Pedata Species (A Nutraceutical Plant) without Liquid Nitrogen for Inter Simple Sequence Repeat Analyses

Comparative analysis indicates a simple protocol for DNA extraction of the aromatic plant Lippia alba

An efficient method of genomic DNA extraction that provides high quality and yield is a crucial pre-requisite and limiting factor in plant genetic analysis. However, pure genomic DNA can be challenging to obtain from some plant species due to their sugar and secondary metabolite contents. Lippia alba is an important aromatic and medicinal plant, chemically characterized by the presence of tannins, flavonoids, anthocyanins, and essential oils, which interfere with the extraction of pure genomic DNA. In this scenario, optimizing the extraction methods and minimizing the effects of these compounds are necessary. This study compares six plant DNA extraction protocols based on the CTAB method. The quality and quantity of DNA samples obtained were determined by physical appearance by electrophoresis in agarose gels and spectrophotometry. The results highlight the difficulty in obtaining pure and clear bands for all tested methods, except for the polyvinylpyrrolidone (PVP)-based protocol created by our team, which was the better option for obtaining high-quality genomic DNA of L. alba. We conclude that adding PVP-40 into DNA extraction buffers can optimize the DNA extraction of L. alba and indicate this protocol for DNA extraction from other aromatic plants.

Improvement and Validation of a Genomic DNA Extraction Method for Human Breastmilk.

The human milk microbiota (HMM) of healthy women can vary substantially, as demonstrated by recent advances in DNA sequencing technology. However, the method used to extract genomic DNA (gDNA) from these samples may impact the observed variations and potentially bias the microbiological reconstruction. Therefore, it is important to use a DNA extraction method that is able to effectively isolate gDNA from a diverse range of microorganisms. In this study, we improved and compared a DNA extraction method for gDNA isolation from human milk (HM) samples to commercial and standard protocols. We evaluated the extracted gDNA using spectrophotometric measurements, gel electrophoresis, and PCR amplifications to assess its quantity, quality, and amplifiability. Additionally, we tested the improved method's ability to isolate amplifiable gDNA from fungi, Gram-positive and Gram-negative bacteria to validate its potential for reconstructing microbiological profiles. The improved DNA extraction method resulted in a higher quality and quantity of the extracted gDNA compared to the commercial and standard protocols and allowed for polymerase chain reaction (PCR) amplification of the V3-V4 regions of the 16S ribosomal gene in all the samples and the ITS-1 region of the fungal 18S ribosomal gene in 95% of the samples. These results suggest that the improved DNA extraction method demonstrates better performance for gDNA extraction from complex samples such as HM.

An efficient, fast and inexpensive method for genomic DNA extraction of fish tissue.

DNA extraction is an essential step for many genetic techniques like PCR and other molecular analyses. Based on the method of extraction and type of tissue used, the quality of extracted DNA for genetic studies varies. An appropriate extraction method is evaluated by the high concentration and purity of DNA. Thus, this study aimed to find a more efficient and effective method of DNA extraction from fish tissues and compare it to commercially available kits. A total of 200 fish tissue samples were extracted using each method and then validated with restriction enzymes and PCR amplification. The result revealed that the mean quantity of the isolated genomic DNA, when measured by Nanodrop for grass and common carp, was estimated at (624.41 ± 34.51) µg/ml and (651.27 ± 46.31) µg/ml, respectively, and the purity of this DNA was about (1.83 ± 0.04) and (1.88 ± 0.03) respectively, as compared to commercial extraction kits. Furthermore, gel electrophoresis was performed on the PCR-ready DNA, and the results were confirmed with restriction enzymes and PCR amplification. Based on results obtained from restriction enzymes and PCR analysis, it was determined that no significant inhibitors existed for the enzymes that were used in molecular biology reactions. As a result, this technique provides an efficient and versatile alternative to the traditional method for obtaining bulk amounts of highly qualified DNA from fish tissue and can be easily used for subsequent analyses such as PCR and several molecular experiments on other fish species.

Expeditious method for genomic dna extraction from mushroom mycelium for downstream applications

Expeditious method for genomic dna extraction from mushroom mycelium for downstream applications

A protocol specialized for microbial DNA extraction from living poplar wood

Microbial DNA extraction is a critical step in metagenomic research. High contents of chemical substances in wood tissues always cause low microbial DNA yield and quality. Up to date, almost no specialized methods involved in microbial DNA extraction from living wood were reported. In this study, an improved protocol (M1) concerning microbial DNA extraction from living poplar wood was developed. We compared microbial DNA yield and quality by M1 with those by other seven methods, including PowerSoil DNA isolation kit (M2), two soil microbial DNA extraction methods (M3 and M4), poplar genomic DNA extraction method from wood (M5), and microbial DNA extraction method from herb stems (M6), isolating bacteria (M7) and isolating fungus (M8). Results showed that M1 yielded much better quality and concentration of microbial DNA than the other methods (M2-M8) from both poplar wetwood and sapwood tissues. Following M1 protocol, 1 g of wetwood sample could yield 272.27 ng/ul (vol=50 ul) pure microbial DNA with the absorption ratios of 1.87 (A260/A230) and 1.66 (A260/A280). For 1 g of sapwood sample, these values were 361.83 ng/ul, 1.85 and 2.24, respectively. These DNA could be stably visualized by agarose gel electrophoresis and amplified by primer sets of bacteria (16S V3-V4, 16S-V4, 16S V4-V5) and fungus (ITS1, ITS2). While, the other seven methods only obtained less or contaminated microbial DNA, which could not be amplified stably by aforementioned primer sets. Our protocol provided an approach for microbial community study in living poplar wood in a more accurate way by molecular biology techniques.

A New Method for Genomic DNA Extraction from Sclerotia of Sclerotium rolfsii Inciting Collar Rot of Lentil for Genomic Investigations

Background: The prevailing methods for the genomic DNA extraction of Sclerotium rolfsii from its mycelium mat is often time consuming and yields poor quality and quantity of genomic DNA owing to presence of the outrageous magnitude of mucilage and polysaccharides. Methods: A new fast track method for DNA isolation from the resting structure (sclerotia) of S. rolfsii by modifying CTAB method deprived of supplementation of proteinase K has been standardized. Result: The protocol produced 500ng DNA from sclerotia with purity vacillating from 1.7 to 1.9 as confirmed by A260/A280 and A260/A230 spectrophotometric documentation. The DNA extracted from sclerotia commissioning protocol was efficaciously used for the further downstream reactions/process like PCR-RAPD, PCR-ISSR and ITS amplification of rDNA-ITS region.

Evaluation of Various Methods for Genomic DNA Extraction from Pure Cultures of Lysis Resistant Campylobacters Isolated from Wild Animals

Evaluation of Various Methods for Genomic DNA Extraction from Pure Cultures of Lysis Resistant Campylobacters Isolated from Wild Animals

Comparative analysis of different fecal DNA extraction methods

The community structure and diversity of the gut microbiota are associated with human diseases. However, the analysis of different community structure might be influenced by experimental approaches such as the quality of DNA extraction. Therefore, evaluating the efficiency of different DNA extraction methods for specific intestinal species is a guideline for obtaining a comprehensive human gut microbial profile, which may assist the in-depth investigation into the structure of the gut microbial community. The aim of this study was to perform a comparative analysis of five different DNA extraction methods. With the aid of qPCR, the efficiency of five DNA extraction kits was evaluated in terms of the purity of the extracted DNA, the DNA concentration, and the abundance of genomic DNA extracted from specific intestinal species. The results showed that the kit Q gave the best extraction results, especially for Gram-positive bacteria such as Lactobacillus and Bifidobacterium. The average DNA concentration of the N kit was lower than that of the Q kit, but there was no significant difference between the two in terms of the purity. Compared to the other three commercial kits (M, PSP, TG), the efficiency of the N kit in extracting the genomic DNA of the specified microorganisms were the least different from those of the Q kit. In contrast, the DNA extracted by the M kit was of higher quality but of lower concentration, and was not very efficient for Gram-positive bacteria. The DNA extracted by the TG and PSP kits was inferior to the other validated kits in terms of the concentration, quality and bacterial abundance. These results provide a basis for the selection of genomic DNA extraction methods in microecological research experiments.

Small-Scale Extraction of Caenorhabditis elegans Genomic DNA.

Genomic DNA extraction from single or a few Caenorhabditis elegans has many downstream applications, including PCR for genotyping lines, cloning, and sequencing. The traditional proteinase K-based methods for genomic DNA extraction from C. elegans take several hours. Commercial extraction kits that effectively break open the C. elegans cuticle and extract genomic DNA are limited. An easy, faster (~15 min), and cost-efficient method of extracting C. elegans genomic DNA that works well for classroom and research applications is reported here. This DNA extraction method is optimized to use single or a few late-larval (L4) or adult nematodes as starting material for obtaining a reliable template to perform PCR. The results indicate that the DNA quality is suitable for amplifying gene targets of different sizes by PCR, permitting genotyping of single or a few animals even at dilutions to one-fiftieth of the genomic DNA from a single adult per reaction. The reported protocols can be reliably used to quickly produce DNA template from a single or a small sample of C. elegans for PCR-based 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.