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A quick and effective in-house method of DNA purification from agarose gel, suitable for sequencing.

ABSTRACT: Sequencing of DNA fragments (e.g., ITS, 16S, 18S, particular genes, and molecular markers) is increasingly required in studies on microbial diversity, microbial genetic population and phylogeny, sequencing of alleles, and searching for SNPs, among others. The cost of obtaining these DNAs, in quantity and quality for sequencing, is high as it involves special kits to recover DNA from gel after PCR, or the cloning and purification of plasmids with commercial kits. Genetic population and other studies require the analyses of many samples, and therefore, the high cost represents an obstacle for carrying out such projects in countries where there is great biodiversity, such as the tropical and subtropical developing countries, where funds are limited. Modifying an already known method for DNA recovery from gel, the first in-house protocol of DNA recovery suitable for direct use in sequencing is presented herein. This protocol is broadly applicable on DNAs from all different living beings, e.g., bacteria, fungi, and plants. Its simplicity, speed, and low cost make this procedure amenable for high-throughput DNA sequencings as required in microbial population studies, development of molecular markers, molecular identification of strains in microbial collections, and others. Recovery of DNA fragments from agarose gel is one of the most common tasks in molecular biology laboratories. Therefore, its potential of applicability of the protocol presented here is enormous.

SUBMITTER: Abraham OJ

PROVIDER: S-EPMC5491447 | biostudies-literature | 2017 Jul

REPOSITORIES: biostudies-literature

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A quick and effective in-house method of DNA purification from agarose gel, suitable for sequencing.

Abraham Obrador-Sánchez José OJ Miguel Tzec-Sima TS Inocencio Higuera-Ciapara HC Blondy Canto-Canché CC

3 Biotech 20170629 3

Sequencing of DNA fragments (e.g., ITS, 16S, 18S, particular genes, and molecular markers) is increasingly required in studies on microbial diversity, microbial genetic population and phylogeny, sequencing of alleles, and searching for SNPs, among others. The cost of obtaining these DNAs, in quantity and quality for sequencing, is high as it involves special kits to recover DNA from gel after PCR, or the cloning and purification of plasmids with commercial kits. Genetic population and other stud ...[more]

PMID: 28664367

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Project description:BackgroundThe recent development of next-generation sequencing DNA marker technologies, such as genotyping-by-sequencing (GBS), generates thousands of informative single nucleotide polymorphism markers in almost any species, regardless of genomic resources. This enables poorly resourced or "orphan" crops/species access to high-density, high-throughput marker platforms which have revolutionised population genetics studies and plant breeding. DNA quality underpins success of GBS methods as the DNA must be amenable to restriction enzyme digestion and sequencing. A barrier to implementing GBS technologies is access to inexpensive, high-throughput extraction methods that yield sequencing-quality genomic DNA (gDNA) from plants. Several high-throughput DNA extraction methods are available, but typically provide low yield or poor quality gDNA, or are costly (US$6-$9/sample) for consumables.ResultsWe modified a non-organic solvent protocol to extract microgram quantities (1-13 μg) of sequencing-quality high molecular weight gDNA inexpensively in 96-well plates from either fresh, freeze-dried or silica gel-dried plant tissue. The protocol was effective for several easy and difficult-to-extract forage, crop, horticultural and common model species including Trifolium, Medicago, Lolium, Secale, Festuca, Malus, Oryza, and Arabidopsis. The extracted DNA was of high molecular weight and digested readily with restriction enzymes. Contrasting with other extraction protocols we assessed, Illumina-based sequencing of GBS libraries developed from this gDNA had very uniform high quality base-calls to the end of sequence reads. Furthermore, DNA extracted using this method has been sequenced successfully with the PacBio long-read platform. The protocol is scalable, readily automated without requirement for fume hoods, requires approximately three hours to process 192 samples (384-576 samples/day), and is inexpensive at US$0.62/sample for consumables.ConclusionsThis versatile, scalable and simple protocol yields high molecular weight genomic DNA suitable for restriction enzyme digestion and next-generation sequencing applications including GBS and long-read sequencing platforms such as PacBio. The low cost, high-throughput, and extraction of high quality gDNA from a range of fresh and dried source plant material makes this method suitable for many sequencing and genotyping applications including large-scale sample screening underpinning breeding programmes.

Project description:Hepatitis B virus (HBV) is a para-retrovirus that reverse transcribes its pregenomic RNA into relaxed circular DNA inside viral nucleocapsids. The number of HBV genomes produced in vitro is typically quantified using commercial silica-membrane-based nucleic acid purification kits to isolate total DNA followed by HBV-specific quantitative PCR (qPCR). However, despite the convenience of commercial kits, this procedure is costly and time-consuming due to multiple centrifugation steps, which produce unnecessary waste. Here, we report a rapid, cost-effective, and environmentally friendly total DNA preparation method. The assay is based on the simple incubation of detergent and proteinase K with cells or cell-free supernatants to permeabilize cells and disrupt viral particles. After heat inactivation and subsequent centrifugation to clear the lysates, DNA samples are directly subjected to qPCR to quantify HBV genomes. As a proof of concept, the assay was developed in 12-well plates to assess intra- and extracellular HBV genome equivalents (GEqs) of stably viral-replicating cell lines (e.g., HepAD38) and HBV-infected HepG2-NTCP cells, both treated with lamivudine (LMV), an HBV replication inhibitor. Viral DNA was also prepared from the serum of patients chronically infected with HBV. To validate the assay, a representative commercial DNA isolation kit was used side-by-side to isolate intra- and extracellular HBV DNA. Both methods yielded comparable amounts of HBV GEqs with comparable LMV 50% efficient concentration (EC50) values. The assay was subsequently adapted to 96- and 384-well microtiter plates using HepAD38 cells. The EC50 values were comparable to those obtained in 12-well plates. In addition, the calculated coefficient of variation, Z' values, and assay window demonstrated high reproducibility and quality. We devised a novel, robust, reproducible, high-throughput microtiter plate DNA preparation method suitable for quantifying HBV GEqs by qPCR analysis. This strategy enables rapid and convenient quantitative analysis of multiple viral DNA samples in parallel to investigate intracellular HBV replication and the secretion of DNA-containing viral particles.

Project description:BackgroundHIV genotyping has had a significant impact on the care and treatment of HIV/AIDS. At a clinical level, the test guides physicians on the choice of treatment regimens. At the surveillance level, it informs policy on consolidated treatment guidelines and microbial resistance control strategies. Until recently, the conventional test has utilized the Sanger sequencing (SS) method. Unlike Next Generation Sequencing (NGS), SS is limited by low data throughput and the inability of detecting low abundant drug-resistant variants. NGS can improve sensitivity and quantitatively identify low-abundance variants; in addition, it has the potential to improve efficiency as well as lowering costs when samples are batched. Despite the NGS benefits, its utilization in clinical drug resistance profiling is faced with mixed reactions. These are largely based on a lack of a consensus regarding the quality control strategy. Nonetheless, transitional views suggest validating the method against the gold-standard SS. Therefore, we present a validation report of an NGS-based in-house HIV genotyping method against the SS method in Uganda.ResultsSince there were no established proficiency test panels for NGS-based HIV genotyping, 15 clinical plasma samples for routine care were utilized. The use of clinical samples allowed for accuracy and precision studies. The workflow involved four main steps; viral RNA extraction, targeted amplicon generation, amplicon sequencing and data analysis. Accuracy of 98% with an average percentage error of 3% was reported for the NGS based assay against the SS platform demonstrating similar performance. The coefficient of variation (CV) findings for both the inter-run and inter-personnel precision showed no variability (CV ≤ 0%) at the relative abundance of ≥ 20%. For both inter-run and inter-personnel, a variation that affected the precision was observed at 1% frequency. Overall, for all the frequencies, CV registered a small range of (0-2%).ConclusionThe NGS-based in-house HIV genotyping method fulfilled the minimum requirements that support its utilization for drug resistance profiling in a clinical setting of a low-income country. For more inclusive quality control studies, well-characterized wet panels need to be established.

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A quick and effective in-house method of DNA purification from agarose gel, suitable for sequencing.

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A quick and effective in-house method of DNA purification from agarose gel, suitable for sequencing.

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