Improved method for genomic DNA extraction for Opuntia Mill. (Cactaceae)

Martínez-González, César Ramiro; Ramírez-Mendoza, Rosario; Jiménez-Ramírez, Jaime; Gallegos-Vázquez, Clemente; Luna‐Vega, Isolda

doi:10.1186/s13007-017-0234-y

Cited by 35 publications

(26 citation statements)

References 32 publications

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“…The use of a guanidine thiocyanate lysis buffer to dissolve mucopolysaccharides was chosen over the use of high salt buffer to preferentially precipitate DNA demonstrated by others (Healey et al 2014) because such differential precipitation is very sensitive and not amenable to processing on a robotic platform. The final step of differentially dissolving the DNA from an undisturbed pellet was taken from method developed for Cactaceae by Martínez-González et al (2017). In most of the samples, traces of co-purified polysaccharides were not visible in the DNA pellet.…”

Section: Discussionmentioning

confidence: 99%

High throughput DNA extraction of legume root nodules for rhizobial metagenomics

Johnson

2019

AMB Expr

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A novel combination of tissue homogenization, cell lysis, and DNA purification techniques was developed for isolating total DNA from whole legume root nodules on an automated robotic system. Silica dehydrated root nodules from soy bean, Glycine max cv. Tara, and mung bean, Vigna radiata cv. Crystal, were homogenized in 96-well plates and were enzymatically and chemically lysed before being loaded into the AutoGenprep 965 for automated phenol–chloroform based DNA isolation. The resulting DNAs were of relatively high concentration, low fragmentation, and free of phenolic and polysaccharide contaminants. In contrast to manual methods, this adaptation of the Autogen Prep automated DNA extraction instrument allows for 100’s to 1000’s of samples to be prepared in a fraction of the time and cost.

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Section: Discussionmentioning

confidence: 99%

High throughput DNA extraction of legume root nodules for rhizobial metagenomics

Johnson

2019

AMB Expr

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“…So far, the CTAB DNA extraction protocol developed by Doyle and Doyle [ 10 ] is one of the most widely used by plant researchers. Several modifications of this protocol have been implemented in order to minimize contamination by other compounds of specific tissues of species [ 7 , 11 , 12 ]. These modifications, apart from being species or tissue-specific and frequently not removing completely interfering compounds, are time-consuming due to many handling steps, and thus are not suitable for high-throughput applications [ 13 , 14 ].…”

Section: Introductionmentioning

confidence: 99%

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

et al. 2020

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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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“…However, with conventional methods, DNA extraction requires the use of toxic solvents and/or multiple and time-consuming steps to remove cellular interferences, such as polysaccharides, polyphenols, and other secondary metabolites, with the risk that DNA isolation can be the major bottleneck in the bioanalytical workflow. As a consequence, fast and cost-efficient DNA extraction protocols that yield high-quality DNA are highly desired in the study of species’ molecular genetics [6–8].…”

Section: Introductionmentioning

confidence: 99%

Development of an innovative and sustainable one-step method for rapid plant DNA isolation for targeted PCR using magnetic ionic liquids

et al. 2019

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Background Nowadays, there is an increasing demand for fast and reliable plant biomolecular analyses. Conventional methods for the isolation of nucleic acids are time-consuming and require multiple and often non-automatable steps to remove cellular interferences, with consequence that sample preparation is the major bottleneck in the bioanalytical workflow. New opportunities have been created by the use of magnetic ionic liquids (MILs) thanks to their affinity for nucleic acids. Results In the present study, a MIL-based magnet-assisted dispersive liquid–liquid microextraction (maDLLME) method was optimized for the extraction of genomic DNA from Arabidopsis thaliana (L.) Heynh leaves. MILs containing different metal centers were tested and the extraction method was optimized in terms of MIL volume and extraction time for purified DNA and crude lysates. The proposed approach yielded good extraction efficiency and is compatible with both quantitative analysis through fluorimetric-based detection and qualitative analysis as PCR amplification of multi and single locus genes. The protocol was successfully applied to a set of plant species and tissues. Conclusions The developed MIL-based maDLLME approach exhibits good enrichment of nucleic acids for extraction of template suitable for targeted PCR; it is very fast, sustainable and potentially automatable thereby representing a powerful tool for screening plants rapidly using DNA-based methods. Electronic supplementary material The online version of this article (10.1186/s13007-019-0408-x) contains supplementary material, which is available to authorized users.

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Improved method for genomic DNA extraction for Opuntia Mill. (Cactaceae)

Cited by 35 publications

References 32 publications

High throughput DNA extraction of legume root nodules for rhizobial metagenomics

High throughput DNA extraction of legume root nodules for rhizobial metagenomics

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

Development of an innovative and sustainable one-step method for rapid plant DNA isolation for targeted PCR using magnetic ionic liquids

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