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

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

Plant Methods ◽

10.1186/s13007-020-00652-y ◽

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

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

Clinical Chemistry ◽

10.1373/clinchem.2012.198945 ◽

2013 ◽

Vol 59 (7) ◽

pp. 1045-1051 ◽

Cited By ~ 32

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.

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

10.1101/2020.02.13.948455 ◽

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.

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

American Journal of Plant Sciences ◽

10.4236/ajps.2014.513217 ◽

2014 ◽

Vol 05 (13) ◽

pp. 2023-2030 ◽

Cited By ~ 4

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

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

10.21203/rs.3.rs-29518/v1 ◽

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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ANALYSIS OF FOUR DIFFERENT METHODS OF DNA EXTRACTION FROM THE TRUE WEEVIL (COLEOPTERA: CURCULIONIDAE)

FLORA AND FAUNA ◽

10.33451/florafauna.v24i1pp135-138 ◽

2018 ◽

Vol 24 (1) ◽

Author(s):

DIVYA SHARMA ◽

DALIP KUMAR ◽

RHITOBAN RAY CHOUDHURY

Keyword(s):

Dna Extraction ◽

Genomic Dna ◽

Extraction Method ◽

Sitophilus Oryzae ◽

Potassium Acetate ◽

Single Specimen ◽

High Quality ◽

Dna Yield ◽

Yield And Quality ◽

Dna Extraction Method

PCR-based markers have been widely used for the analysis of genetic diversity and to avoid ambiguity, molecular characterization is very effective tool for accurate discrimination and identification of a species in insects. Because these studies require analysis of large number of samples, a DNA extraction method that is fast, inexpensive and yields high quality DNA from the preserved samples, needs to be evaluated. A comparative analysis of four methods for DNA extraction from a single specimen of rice weevil, Sitophilus oryzae preserved in 90% alcohol has been communicated. Significantly higher DNA yields were obtained by using SDS-Potassium acetate method followed by CTAB, DNA XPress and Bioline Isolate II genomic DNA kit. Maximum purity (A260/A280- 1.8) was obtained with Bioline Isolate II genomic DNA kit method. The Absorbance ratio was appreciably low with DNA Xpress kit showing the presence of proteins. Bioline Isolate II genomic DNA kit was time efficient and yielded good quality DNA but at a high cost. Based on DNA yield and quality, these evaluations provide a guide for choosing Bioline Isolate II genomic DNA kit method of DNA extraction for rice weevils and optimizing the extraction conditions for rice weevils.

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DNA Extraction from Bronchial Aspirates for Molecular Cytology: Which Method to Take?

Analytical Cellular Pathology ◽

10.1155/2003/354796 ◽

2003 ◽

Vol 25 (2) ◽

pp. 83-88 ◽

Cited By ~ 7

Author(s):

Hans Jürgen Grote ◽

Viola Schmiemann ◽

Mario Sarbia ◽

Alfred Böcking

Keyword(s):

Success Rate ◽

Dna Extraction ◽

Extraction Method ◽

Globin Gene ◽

Extraction Methods ◽

High Quality ◽

High Molecular Weight Dna ◽

Dna Yield ◽

Extraction Protocol ◽

Dna Extraction Method

Objective: To date, there are only few systematic reports on the quality of DNA extracted from routine diagnostic cytologic specimens. It was the aim of the present study to evaluate the ability of 50% ethanol/2% carbowax (Saccomanno fixative) to preserve bronchial secretions with high quality genomic DNA as well as to compare different DNA extraction methods.Methods: DNA was extracted from 45 bronchial aspirates by four different extraction protocols. Beside DNA yield, DNA quality with regard to purity, integrity, and PCR success rate were investigated.Results: No fragmentation of sample DNA due to the fixative was detected. It was preserved as high molecular weight DNA. DNA yield, purity, and integrity were dependent on the DNA extraction method to some extend. Irrespective of the DNA extraction method the PCR success rate for amplification of β‐globin gene fragments (268, 536, and 989 bp) was 100%. Conclusions: A fixative containing 50% ethanol/2% carbowax preserves high quality DNA which is well suited for PCR‐based assays regardless of the extraction protocol used. The selection of the DNA extraction protocol has to be adjusted to the circumstances of application.

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