Extraction of high purity genomic DNA from pine for use in a high-throughput Genotyping Platform

Telfer, Emily; Graham, Natalie; Stanbra, Lisa; Manley, T. R.; Wilcox, Phillip

doi:10.1186/1179-5395-43-3

Cited by 24 publications

(20 citation statements)

References 13 publications

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“…Thechaotropic agents (for instance guanidinium thiocyanate) used in this method would have played a role to remove DNA-binding proteins, allowing for better absorption in the spin columns (Boom et al 1989). The results confirmed that the use of cell lysis buffer and following elution over a column could greatly enhance the amount of the extracted pure mDNAs, which was previously reported also by other researchers (Poh and Gan 2014;Telfer et al 2013). In addition to the technical efficiency, the SCB protocol is significantly more cost-effective (∼ US$ 1 per reaction) compared with the commercial kits (∼ US$ 7 per reaction), which makes this method suitable for molecular biology studies, such as PCR amplifications.…”

Section: Discussionsupporting

confidence: 90%

Rapid and economical protocols for genomic and metagenomic DNA extraction from oak (Quercus brantii Lindl.)

Ahmadi

Kowsari

Azadfar

et al. 2018

Annals of Forest Science

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& Key message Two new efficient, fast and low-cost metagenomic DNA extraction methods were developed for different Persian oak tissues (leaf, stem, root, and rhizospheric) and soil samples. & Context The new "omics" studies on the genus Quercus are of importance to help finding efficient strategies for overcoming environmental challenges, and to do this, presence of efficient DNA extraction protocols for different Quercus species are very critical. & Aims The objective of the present study was to develop new efficient methods for extraction of metagenomic DNA (mDNA) from of Persian oak (Quercus brantii Lindl.) tissues. & Methods The efficiency of two newly developed mDNA extraction methods, including indirect SDS-based (ISB or concentrate method) and one spin column-based method (SCB) were compared to that of two classical direct methods, including CTABbased and SDS-based methods, and two commercial mDNA extraction kits. & Results The maximum average yield of mDNA for all samples (leaf, stem, root, bulk, and rhizospheric soils) was obtained by SCB (258 ng/μl) and ISB (189 ng/μl) methods, respectively. Successful PCR amplification for 16SrRNA and ITS sequence was consistently observed for ISB, SCB, and kit-extracted mDNAs, which confirmed the high purity of mDNA extracted by these methods. The new methods showed more than 96% quantitative PCR efficiency, and partial restriction digestion and metagenomic library construction confirmed the high efficiency of the newly developed methods. & Conclusion It could be concluded that two new protocols enhanced efficiency (yield, purity, and cost) of mDNA extraction from different tissues of Persian oak.

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Section: Discussionsupporting

confidence: 90%

Rapid and economical protocols for genomic and metagenomic DNA extraction from oak (Quercus brantii Lindl.)

Ahmadi

Kowsari

Azadfar

et al. 2018

Annals of Forest Science

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“…Considering the high level of PCR sensitivity obtained with genomic DNA in these assays, the low detection rates observed for C. minus from pine needles may indicate that the DNA extraction method used may not be efficient enough for pathogen detection from P. radiata. Obtaining high-quality DNA from pine needle samples can be difficult due to high amounts of co-contaminants (Telfer et al 2013). Comparisons of DNA extraction methods have been performed to identify the best method for obtaining high-quality DNA for PCR from P. radiata (Ioos et al 2010;Telfer et al 2013), and these methods should be tested for improved detection of C. minus from infected pine needles.…”

Section: Discussionmentioning

confidence: 99%

Molecular tools for differentiating Cyclaneusma minus morphotypes and assessing their distribution in Pinus radiata forests in New Zealand

Hunter

Glen

McDougal

2016

N.Z. j. of For. Sci.

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Background: Cyclaneusma needle cast (CNC) is a pine disease caused by the ascomycetous fungus Cyclaneusma minus (Butin) DiCosmo, Peredo and Minter. The pathogen occurs worldwide but is of particular significance in New Zealand where it infects Pinus radiata D. Don plantations. There are two morphological types of C. minus, termed C. minus 'simile' and C. minus 'verum', recently shown by multigene phylogenetic analysis to belong to distinct clades and therefore proposed to be two separate species. It is currently unknown whether one or both of these molecular operational taxonomic units (MOTUs) are responsible for CNC. Methods: In this study, DNA analysis of the rDNA internal transcribed spacer (ITS) region was carried out on 120 isolates of C. minus collected in New Zealand since 1969 to distinguish C. minus simile from C. minus verum. Specific primers for C. minus simile and C. minus verum were developed for molecular differentiation from pure cultures and direct amplification from infected needles. Using these specific primers, the distribution of C. minus simile and verum was determined from isolates collected throughout New Zealand. Results: The C. minus simile and C. minus verum primers developed were specific to these morphotypes only and were successfully used to identify simile and verum MOTUs from culture. Morphological typing of cultures was consistent with specific primer results in only 56% of isolates tested, demonstrating the high level of skill and experience required for accurate morphotyping from culture alone. C. minus simile was more common throughout New Zealand including regions where CNC is known to be more prevalent. From a collection of 120 isolates, 89% of North Island isolates and 59% of the South Island isolates were C. minus simile. The C. minus simile and verum specific primers were used to detect Cyclaneusma DNA in symptomatic P. radiata needles. C. minus simile was detected in 39% of the needle samples and C. minus verum was only detected in 11%. However, Cyclaneusma DNA was not detected by PCR in 50% of the sampled needles. Conclusions: The molecular methods developed for differentiation of C. minus simile and C. minus verum can be used to rapidly identify these morphotypes from cultures or from infected needles, which reduces the time otherwise required for morphological identification. Analysis using these tools for characterisation of C. minus sensu lato, isolated from P. radiata plantations in New Zealand since 1969, revealed that C. minus simile was more common than C. minus verum.

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“…DNA extractions from leaves were performed using the NucleoSpin® Plant II 96 kit (Macherey Nagel, Duren, Germany; Telfer et al, 2013) implemented on a Tecan Genesis 200RMP (Tecan, Maennedorf, Switzerland) liquidhandling robot. In short, two 1 cm² fresh or frozen leaves were homogenized in a 2 ml microcentrifuge tube using the TissueLyser (Qiagen, Venlo, Netherlands).…”

Section: Plant Materials and Dna Extractionmentioning

confidence: 99%

Microsatellite-based DNA Fingerprinting of Selected Grapevine Cultivars

Heerden

Burger

Prins

2018

SAJEV

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Extraction of high purity genomic DNA from pine for use in a high-throughput Genotyping Platform

Cited by 24 publications

References 13 publications

Rapid and economical protocols for genomic and metagenomic DNA extraction from oak (Quercus brantii Lindl.)

Rapid and economical protocols for genomic and metagenomic DNA extraction from oak (Quercus brantii Lindl.)

Molecular tools for differentiating Cyclaneusma minus morphotypes and assessing their distribution in Pinus radiata forests in New Zealand

Microsatellite-based DNA Fingerprinting of Selected Grapevine Cultivars

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