scholarly journals Comparative evaluation of different molecular methods for DNA extraction from individual Teladorsagia circumcincta nematodes

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
S. Sloan ◽  
C. J. Jenvey ◽  
D. Piedrafita ◽  
S. Preston ◽  
M. J. Stear
Keyword(s):  
Dna Extraction ◽  
Comparative Evaluation ◽  
Processing Time ◽  
High Quality ◽  
Dna Concentration ◽  
Teladorsagia Circumcincta ◽  
Dna Extraction Method ◽  
Fast Processing ◽  
Nematode Cuticle ◽  
Pooled Samples

Abstract Background The purpose of this study was to develop a reliable DNA extraction protocol to use on individual Teladorsagia circumcincta nematode specimens to produce high quality DNA for genome sequencing and phylogenetic analysis. Pooled samples have been critical in providing the groundwork for T. circumcincta genome construction, but there is currently no standard method for extracting high-quality DNA from individual nematodes. 11 extraction kits were compared based on DNA quality, yield, and processing time. Results 11 extraction protocols were compared, and the concentration and purity of the extracted DNA was quantified. Median DNA concentration among all methods measured on NanoDrop 2000™ ranged between 0.45–11.5 ng/μL, and on Qubit™ ranged between undetectable – 0.962 ng/μL. Median A260/280 ranged between 0.505–3.925, and median A260/230 ranged − 0.005 – 1.545. Larval exsheathment to remove the nematode cuticle negatively impacted DNA concentration and purity. Conclusions A Schistosoma sp. DNA extraction method was determined as most suitable for individual T. circumcincta nematode specimens due to its resulting DNA concentration, purity, and relatively fast processing time.

scholarly journals SILEX: A fast and inexpensive high-quality DNA extraction method suitable for multiple sequencing platforms and recalcitrant plant species

2020 ◽  
Author(s):  
santiago vilanova ◽  
David Alonso ◽  
Pietro Gramazio ◽  
Mariola Plazas ◽  
Edgar Garcia Fortea ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Next Generation Sequencing ◽  
Dna Extraction ◽  
High Throughput ◽  
Extraction Method ◽  
High Quality ◽  
Extraction Protocol ◽  
Dna Extraction Method ◽  
Sequencing Platforms ◽  
Generation Sequencing

Abstract Background The use of sequencing and genotyping platforms has undergone dramatic improvements, enabling the generation of a wealth of genomic information. Despite this progress, the availability of high-quality genomic DNA (gDNA) in sufficient concentrations is often a main limitation, especially for third-generation sequencing platforms. A variety of DNA extraction methods and commercial kits are available. However, many of these are costly and frequently give either low yield or low-quality DNA, inappropriate for next generation sequencing (NGS) platforms. Here, we describe a fast and inexpensive DNA extraction method (SILEX) applicable to a wide range of plant species and tissues. Results SILEX is a high-throughput DNA extraction protocol, based on the standard CTAB method with a DNA silica matrix recovery, which allows obtaining NGS-quality high molecular weight genomic plant DNA free of inhibitory compounds. SILEX was compared with a standard CTAB extraction protocol and a common commercial extraction kit in a variety of species, including recalcitrant ones, from different families. In comparison with the other methods, SILEX yielded DNA in higher concentrations and of higher quality. Manual extraction of 48 samples can be done in 96 min by one person at a cost of 0.12 €/sample of reagents and consumables. Hundreds of tomato gDNA samples obtained with either SILEX or the commercial kit were successfully genotyped with Single Primer Enrichment Technology (SPET) with the Illumina HiSeq 2500 platform. Furthermore, DNA extracted from Solanum elaeagnifolium using this protocol was assessed by Pulsed-field gel electrophoresis (PFGE), obtaining a suitable size ranges for most sequencing platforms that required high-molecular-weight DNA such as Nanopore or PacBio. Conclusions A high-throughput, fast and inexpensive DNA extraction protocol was developed and validated for a wide variety of plants and tissues. SILEX offers an easy, scalable, efficient and inexpensive way to extract DNA for various next-generation sequencing applications including SPET and Nanopore among others.

scholarly journals SILEX: a fast and inexpensive high-quality DNA extraction method suitable for multiple sequencing platforms and recalcitrant plant species

Plant Methods ◽  
2020 ◽  
Vol 16 (1) ◽  
Author(s):  
Santiago Vilanova ◽  
David Alonso ◽  
Pietro Gramazio ◽  
Mariola Plazas ◽  
Edgar García-Fortea ◽  
...  
Keyword(s):  
Dna Extraction ◽  
Plant Species ◽  
Extraction Method ◽  
High Quality ◽  
Dna Extraction Method ◽  
Sequencing Platforms

scholarly journals Cost-Effective and Scalable DNA Extraction Method from Dried Blood Spots

2013 ◽  
Vol 59 (7) ◽  
pp. 1045-1051 ◽  
Author(s):  
Carlos A Saavedra-Matiz ◽  
Jason T Isabelle ◽  
Chad K Biski ◽  
Salvatore J Duva ◽  
Melissa L Sweeney ◽  
...  
Keyword(s):  
Dna Extraction ◽  
High Throughput ◽  
Extraction Method ◽  
Low Cost ◽  
Melting Curve Analysis ◽  
Molecular Testing ◽  
High Quality ◽  
Congenital Diseases ◽  
Cell Counts ◽  
Dna Extraction Method

BACKGROUND Dried blood spot (DBS) samples have been widely used in newborn screening (NBS) for the early identification of disease to facilitate the presymptomatic treatment of congenital diseases in newborns. As molecular genetics knowledge and technology progresses, there is an increased demand on NBS programs for molecular testing and a need to establish reliable, low-cost methods to perform those analyses. Here we report a flexible, cost-efficient, high-throughput DNA extraction method from DBS adaptable to small- and large-scale screening settings. METHODS Genomic DNA (g.DNA) was extracted from single 3-mm diameter DBS by the sequential use of red cell lysis, detergent-alkaline, and acid-neutralizing buffers routinely used in whole blood and plant tissue DNA extractions. We performed PCR amplification of several genomic regions using standard PCR conditions and detection methods (agarose gel, melting-curve analysis, TaqMan-based assays). Amplicons were confirmed by BigDye® Terminator cycle sequencing and compared with reference sequences. RESULTS High-quality g.DNA was extracted from hundreds of DBS, as proven by mutation detection of several human genes on multiple platforms. Manual and automated extraction protocols were validated. Quantification of g.DNA by Oligreen® fluorescent nucleic acid stain demonstrated a normal population distribution closely corresponding with white blood cell counts detected in newborn populations. CONCLUSIONS High-quality, amplifiable g.DNA is extractable from DBSs. Our method is adaptable, reliable, and scalable to low- and high-throughput NBS at low cost ($0.10/sample). This method is routinely used for molecular testing in the New York State NBS program.

scholarly journals A one-tube method for rapid and reliable plant genomic DNA isolation for PCR analysis

2020 ◽  
Author(s):  
Wei Hu ◽  
J. Clark Lagarias
Keyword(s):  
Dna Extraction ◽  
Genomic Dna ◽  
Extraction Method ◽  
Low Cost ◽  
Plant Molecular Biology ◽  
Pcr Analysis ◽  
High Quality ◽  
Plant Dna ◽  
Genomic Dna Isolation ◽  
Dna Extraction Method

AbstractBackgroundConsistent isolation of high quality plant genomic DNA is a prerequisite for successful PCR analysis. Time consumption, ease of operation and procedure cost are important secondary considerations for selecting an effective DNA extraction method. The simple, reliable and rapid DNA extraction method developed by Edwards and colleagues in 1991 [1] has proven to be the gold standard.ResultsThrough modification of the Edwards method of extraction, we have developed a one-tube protocol that greatly improves the efficiency of plant DNA extraction and reduces the potential for sample contamination while simultaneously yielding high quality DNA suitable for PCR analysis. We further show that DNA extracts prepared with this method are stable at room temperature for at least three months.ConclusionThe one-tube extraction method yields high quality plant DNA with improved efficiency while greatly minimizing the potential for cross contamination. This low-cost and environment-friendly method is widely applicable for plant molecular biology research.

scholarly journals High Quality DNA Obtained from a Single Seed of <i>Vitis vinifera</i> L. Using Rapid DNA Extraction Method

2014 ◽  
Vol 05 (13) ◽  
pp. 2023-2030 ◽  
Author(s):  
Ajith Samantha Rathnayake ◽  
Josep Allué ◽  
Mercè Llugany ◽  
Anna Puig-Pujol ◽  
Kshanika Hirimburegama ◽  
...  
Keyword(s):  
Vitis Vinifera ◽  
Dna Extraction ◽  
Extraction Method ◽  
High Quality ◽  
Single Seed ◽  
Dna Extraction Method

scholarly journals A Modified SDS-Based DNA Extraction Method for High Quality Environmental DNA from Seafloor Environments

2016 ◽  
Vol 07 ◽  
Author(s):  
Vengadesh Perumal Natarajan ◽  
Xinxu Zhang ◽  
Yuki Morono ◽  
Fumio Inagaki ◽  
Fengping Wang
Keyword(s):  
Dna Extraction ◽  
Extraction Method ◽  
Environmental Dna ◽  
High Quality ◽  
Dna Extraction Method

scholarly journals Comparison of DNA Concentration and Purity of Animal Blood Extracted using Different DNA Extraction Kits

2018 ◽  
Vol 2 ◽  
Author(s):  
Mohamed Sabri Esa ◽  
Nur Huda Faujan ◽  
Haitham Abdullah Rajab ◽  
Maryam Mohamed Rehan ◽  
Norlelawati Arifin ◽  
...  
Keyword(s):  
Dna Extraction ◽  
Genomic Dna ◽  
Extraction Method ◽  
Bos Taurus ◽  
Dna Purification ◽  
Chain Reaction ◽  
Animal Blood ◽  
Dna Concentration ◽  
Dna Extraction Method ◽  
Polymerase Chain

The efficiency of DNA extraction from whole blood using appropriate method is very important for molecular analysis. Therefore, the aim of this study was to compare the purity and concentration of DNA extraction method from bovine (Bos taurus), chicken (Gallus gallus), and porcine (Sus scrofa) blood. The DNA of blood samples was extracted using three types of kit, namely InnuPREP Blood DNA Mini Kit, Wizard Genomic DNA Purification Kit, and QIAamp DNA Blood Mini Kit. The results showed that blood DNA extracted using QIAamp DNA Blood Mini Kit was found to be the most effective and consistently produced high concentrated and pure DNA for three animal samples. The purity of DNA ranged from 1.73 ± 0.05 Å to 1.94 ± 0.21 Å and the range of blood DNA concentration extracted using the QIAamp DNA were between 13.73 ± 2.11 and 25.01 ± 2.08 ng/?l. However, the blood DNA of porcine was not successfully extracted using InnuPREP Blood DNA Mini Kit and Wizard® Genomic DNA Purification Kit. These results were very crucial for the subsequent use of amplification using polymerase chain reaction (PCR) and to facilitate accurate detection in further analysis.

scholarly journals SILEX: A fast and inexpensive high-quality DNA extraction method suitable for multiple sequencing platforms and recalcitrant plant species

2020 ◽  
Author(s):  
santiago vilanova ◽  
David Alonso ◽  
Pietro Gramazio ◽  
Mariola Plazas ◽  
Paola Ferrante ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Next Generation Sequencing ◽  
Dna Extraction ◽  
High Throughput ◽  
Extraction Method ◽  
High Quality ◽  
Extraction Protocol ◽  
Dna Extraction Method ◽  
Sequencing Platforms ◽  
Generation Sequencing

Abstract Background: The use of sequencing and genotyping platforms has undergone dramatic improvements, enabling the generation of a wealth of genomic information. Despite this progress, the availability of high-quality genomic DNA (gDNA) in sufficient concentrations is often a main limitation, especially for third-generation sequencing platforms. A variety of DNA extraction methods and commercial kits are available. However, many of these are costly and frequently give either low yield or low-quality DNA, inappropriate for next generation sequencing (NGS) platforms. Here, we describe a fast and inexpensive DNA extraction method (SILEX) applicable to a wide range of plant species and tissues. Results: SILEX is a high-throughput DNA extraction protocol, based on the standard CTAB method with a DNA silica matrix recovery, which allows obtaining NGS-quality high molecular weight genomic plant DNA free of inhibitory compounds. SILEX was compared with a standard CTAB extraction protocol and a common commercial extraction kit in a variety of species, including recalcitrant ones, from different families. In comparison with the other methods, SILEX yielded DNA in higher concentrations and of higher quality. Manual extraction of 48 samples can be done in 96 min by one person at a cost of 0.12 €/sample of reagents and consumables. Hundreds of tomato gDNA samples obtained with either SILEX or the commercial kit were successfully genotyped with Single Primer Enrichment Technology (SPET) with the Illumina HiSeq 2500 platform. Furthermore, DNA extracted from Solanum elaeagnifolium using this protocol was assessed by Pulsed-field gel electrophoresis (PFGE), obtaining a suitable size ranges for most sequencing platforms that required high-molecular-weight DNA such as Nanopore or PacBio. Conclusions: A high-throughput, fast and inexpensive DNA extraction protocol was developed and validated for a wide variety of plants and tissues. SILEX offers an easy, scalable, efficient and inexpensive way to extract DNA for various next-generation sequencing applications including SPET and Nanopore among others.

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