Isolation and Purification of DNA from Complicated Biological Samples

Kalendar, Ruslan; Boronnikova, Svetlana; Seppänen, Mervi

doi:10.1007/978-1-0716-0997-2_3

Cited by 39 publications

(27 citation statements)

References 31 publications

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“…For the study, the needles samples were collected individually from 25-30 trees in each of ten populations of P. sylvestris. DNA was isolated according to the procedure for complex biological samples [23]. The weighed amount of the needles made up 20 mg. NanoDrop 2000 Spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) was used to determine the concentration and quality of DNA.…”

Section: Methodsmentioning

confidence: 99%

Genetic Structure, Differentiation and Originality of Pinus sylvestris L. Populations in the East of the East European Plain

Vasilyeva

Chertov

Nechaeva

et al. 2021

Forests

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In order to carry out activities aimed at conservation and rational use of forest resources; it is necessary to study the main forest-forming plant species in detail. Scots pine (Pinus sylvestris L., Pinaceae) is mainly found in the boreal forests of Eurasia and is not so often encountered in the east of the East European Plain. The aim of the study was to study the genetic diversity, structure and differentiation of Scots pine populations in the east of the East European Plain. We studied ten populations of P. sylvestris using the Inter Simple Sequence Repeats (ISSR)-based DNA polymorphism detection method. Natural populations are demonstrated by relatively high rates of genetic diversity (He = 0.167; ne = 1.279; I = 0.253). At the same time, there is a tendency for a decrease in the genetic diversity of the studied populations of P. sylvestris from west to east. Analysis of the genetic structure shows that the studied populations are highly differentiated (GST = 0.439), the intrapopulation component accounts for about 56% of the genetic diversity. Using various algorithms for determining the spatial genetic structure, it is found that the studied populations form two groups of populations in accordance with geographic location. With the help of a genetic originality coefficient, populations with specific and typical gene pools are identified. They are recommended as sources of genetic diversity and reserves for the conservation of genetic resources of the species.

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Section: Methodsmentioning

confidence: 99%

Genetic Structure, Differentiation and Originality of Pinus sylvestris L. Populations in the East of the East European Plain

Vasilyeva

Chertov

Nechaeva

et al. 2021

Forests

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“…Leaves for DNA isolation were collected from 12-day-old plants. Genomic DNA was extracted using a CTAB-based protocol and treated with RNase A (doi: 10.17504/protocols.io.mghc3t6) ( Kalendar et al, 2021a ). DNA samples were diluted in 1X TE buffer (10 mM Tris–HCl pH 7.5, 1 mM EDTA) and DNA quality was verified using a Nanodrop spectrophotometer (Thermo Fisher Scientific) and gel electrophoresis.…”

Section: Methodsmentioning

confidence: 99%

Palindromic Sequence-Targeted (PST) PCR, Version 2: An Advanced Method for High-Throughput Targeted Gene Characterization and Transposon Display

2021

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Genome walking (GW), a strategy for capturing previously unsequenced DNA fragments that are in proximity to a known sequence tag, is currently predominantly based on PCR. Recently developed PCR-based methods allow for combining of sequence-specific primers with designed capturing primers capable of annealing to unknown DNA targets, thereby offering the rapidity and effectiveness of PCR. This study presents a methodological improvement to the previously described GW technique known as palindromic sequence-targeted PCR (PST-PCR). Like PST-PCR, this new method (called PST-PCR v.2) relies on targeting of capturing primers to palindromic sequences arbitrarily present in natural DNA templates. PST-PCR v.2 consists of two rounds of PCR. The first round uses a combination of one sequence-specific primer with one capturing (PST) primer. The second round uses a combination of a single (preferred) or two universal primers; one anneals to a 5′ tail attached to the sequence-specific primer and the other anneals to a different 5′ tail attached to the PST primer. The key advantage of PST-PCR v.2 is the convenience of using a single universal primer with invariable sequences in GW processes involving various templates. The entire procedure takes approximately 2–3 h to produce the amplified PCR fragment, which contains a portion of a template flanked by the sequence-specific and capturing primers. PST-PCR v.2 is highly suitable for simultaneous work with multiple samples. For this reason, PST-PCR v.2 can be applied beyond the classical task of GW for studies in population genetics, in which PST-PCR v.2 is a preferred alternative to amplified fragment length polymorphism (AFLP) or next-generation sequencing. Furthermore, the conditions for PST-PCR v.2 are easier to optimize, as only one sequence-specific primer is used. This reduces non-specific random amplified polymorphic DNA (RAPD)-like amplification and formation of non-templated amplification. Importantly, akin to the previous version, PST-PCR v.2 is not sensitive to template DNA sequence complexity or quality. This study illustrates the utility of PST-PCR v.2 for transposon display (TD), which is a method to characterize inter- or intra-specific variability related to transposon integration sites. The Ac transposon sequence in the maize (Zea mays) genome was used as a sequence tag during the TD procedure to characterize the Ac integration sites.

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“…Briefly, 100 mg of ground and homogenized sample was transferred to a 2 mL Eppendorf tube followed by the addition of 1 mL of CTAB extraction buffer CTAB extraction buffer (2%, 2M NaCl, 10 mM Na 3 EDTA, 100 mM HEPES, pH 5.3) with proteinase K (30 μg/μl) (http://primerdigital.com/dna.html). A detailed protocol for DNA isolation was followed as described in Kalendar et al [4]. The DNA pellets were dissolved with 1 ×TE buffer (1 mM EDTA, 10 mM Tris-HCl, pH 8.0) with RNase A (1 ng/ml).…”

Section: Methodsmentioning

confidence: 99%

Express Method for Species idenTification of Livestock Products Based on Multiplex PCR

Kalendar¹,

Bektayev²

2020

Eurasian Journal of Applied Biotechnology

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Food falsification has direct impact on public health, fair-trades and wildlife. A test system for simultaneous detecting DNA of Bos taurus/Ovis aries, Equus cabalus, Sus scrofa, Canis lupus, Mus musculus/Rattus rattus species, and Homo sapiens in livestock products was developed. Species-specific primers sets were designed targeting SINE repeat elements. Screening of target species in commercial sausages reflected its application to detect target species in process foods. The assay was tested to detect at least 0.01 pg DNA under suspected meats in sausages. The high sensitivity and specificity of species identification proposed method was shown. The possibility of using the developed test system for assessing the qualitative composition of various food products was demonstrated.

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Isolation and Purification of DNA from Complicated Biological Samples

Cited by 39 publications

References 31 publications

Genetic Structure, Differentiation and Originality of Pinus sylvestris L. Populations in the East of the East European Plain

Genetic Structure, Differentiation and Originality of Pinus sylvestris L. Populations in the East of the East European Plain

Palindromic Sequence-Targeted (PST) PCR, Version 2: An Advanced Method for High-Throughput Targeted Gene Characterization and Transposon Display

Express Method for Species idenTification of Livestock Products Based on Multiplex PCR

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