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1.
Most steps in plant nucleic acid isolation are easily adapted to 96-well format; however, tissue disruption typically is performed
on samples individually and often is the rate-limiting step in sample processing. We have found that DNA and RNA isolation
from Arabidopsis tissue can be carried out in 96-well format using a paint shaker fitted with an adapter for tissue disruption. 相似文献
2.
Simon J. McIlroy Kate Porter Robert J. Seviour Daniel Tillett 《Antonie van Leeuwenhoek》2009,96(4):593-605
Critical to most studies in molecular microbial ecology is the application of DNA/RNA extraction methods which can reveal
the true level of population biodiversity present in samples from the community under investigation. Activated sludge communities
have been studied extensively using molecular methods, but rarely have the nucleic acid isolation methods applied been assessed
for their ability to achieve this. This study compares eight published RNA and DNA extraction protocols and one commercially
available DNA isolation kit for their capacity to provide high quality nucleic acids that reflect the community composition.
Each method was assessed on the basis of nucleic acid yield, purity and integrity, and the ability to provide PCR amplifiable
RNA and DNA from known marker populations that varied in their resistance to nucleic acid extraction. Only three consistently
provided DNA from each of the marker populations known to be present in the samples from fluorescence in situ hybridisation
analysis. The failure of the other methods emphasises the need to validate all DNA/RNA extraction protocols. It is recommended
that several validated extraction methods be used and the extracts pooled to further minimise any risk of bias. 相似文献
3.
TNA (alpha-L-threose nucleic acids) is potentially a natural nucleic acid, that might have acted as an evolutionary alternative of RNA. We determined the catalytic activity of hammerhead ribozymes containing a threofuranosyl-modified nucleoside at position U4 and U7, and compared these results with those obtained from HNA (hexitol nucleic acids) insertion into the same ribozyme. Our experiments showed that, although the threofuranosyl-modified ribozymes still cleave the substrate strand, cleavage activity is highly decreased. It, therefore, seems that TNA can play a functional role in the RNA world, but only to a limited extent. 相似文献
4.
Systematic investigation into the chemical etiology of ribose has led to the discovery of glycerol nucleic acid (GNA) and
threose nucleic acid (TNA) as possible progenitor candidates of RNA in the origins of life. Coupled with their chemical simplicity,
polymers for both systems are capable of forming stable Watson-Crick antiparallel duplex structures with themselves and RNA,
thereby providing a mechanism for the transfer of genetic information between successive genetic systems. Investigation into
whether both polymers arose independently or descended from a common evolutionary pathway would provide additional constraints
on models that describe the emergence of a hypothetical RNA world. Here we show by thermal denaturation that complementary
GNA and TNA mixed sequence polymers are unable, even after prolonged incubation times, to adopt stable helical structures
by intersystem cross-pairing. This experimental observation suggests that GNA and TNA, whose structures derive from one another,
were not consecutive polymers in the same evolutionary pathway to RNA.
Reviewing
Editor: Dr. Niles Lehman 相似文献
5.
Vuosku J Jaakola L Jokipii S Karppinen K Kämäräinen T Pelkonen VP Jokela A Sarjala T Hohtola A Häggman H 《Molecular biotechnology》2004,27(3):209-215
An automated nucleic acid extraction procedure with magnetic particles originally designed for isolation of deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) from animal tissues was tested for plant material. We isolated genomic DNA and total RNA from taxonomically diverse plant species representing conifers (Scots pine), broad-leaved trees (silver birch and hybrid aspen), dwarf shrubs (bilberry), and both monocotyledonous (regal lily) and dicotyledonous (Saint John's wort, round-leaved sundew, and tobacco) herbaceous plants. Buffers developed for DNA extraction were successfully used in addition to manufacturer's extraction kits. The quality of RNA was appropriate for many applications, but the quality of DNA was not always sufficient for polymerase chain reaction (PCR) amplification. However, we could strikingly improve the quality by eliminating the adherent compounds during the extraction or later in the PCR phase. Our results show that the use of the procedure could be extended to diverse plant species. This procedure is especially suitable for small sample sizes and for simultaneous processing of many samples enabling large-scale plant applications in population genetics, or in the screening of putative transgenic plants. 相似文献
6.
Yajun Wang Xiaolin Liu Mouhamad Shehabat Nicholas Chim John
C Chaput 《Nucleic acids research》2021,49(20):11438
Functional nucleic acids lose activity when their sequence is prepared in the backbone architecture of a different genetic polymer. The only known exception to this rule is a subset of aptamers whose binding mechanism involves G-quadruplex formation. We refer to such examples as transliteration—a synthetic biology concept describing cases in which the phenotype of a nucleic acid molecule is retained when the genotype is written in a different genetic language. Here, we extend the concept of transliteration to include nucleic acid enzymes (XNAzymes) that mediate site-specific cleavage of an RNA substrate. We show that an in vitro selected 2′-fluoroarabino nucleic acid (FANA) enzyme retains catalytic activity when its sequence is prepared as α-l-threofuranosyl nucleic acid (TNA), and vice versa, a TNA enzyme that remains functional when its sequence is prepared as FANA. Structure probing with DMS supports the hypothesis that FANA and TNA enzymes having the same primary sequence can adopt similarly folded tertiary structures. These findings provide new insight into the sequence-structure-function paradigm governing biopolymer folding. 相似文献
7.
This report deals with the quantitative extraction of total nucleic acid (TNA) containing undegraded RNA from the slime mold Physarum polycephalum. With the use of a three-step phenol extraction technique, approx. 95 % of the nucleic acid optical density and 90 % of the 3H-uridine incorporated radioactivity were routinely recovered in the extracts. With the use of this technique it was shown that (1) the TNA mg dry wt of the mold did not change throughout the mitotic cycle, even though the dry wt doubled; this indicates a continual net synthesis of nucleic acid throughout the cycle; (2) the relative proportions of the various nucleic acid components did not change significantly during the cycle and were found to be DNA, 6 %; rRNA, 82 %; and sRNA, 12 %; (3) RNA molecules with mol wts of 4.1 m and 1.9 m, which exhibit properties of rRNA precursors were found in plasmodia labeled for 20 min with 3H-uridine. Furthermore, there appears to be an RNA fraction, found only in nucleic acid preparations presumably enriched in nuclear RNA components, which is heat-labile, does not enter 2.6 % acrylamide gels during 4 h of electrophoresis, and has a uridine/methyl ratio different from the presumed rRNA precursors and mature rRNA. 相似文献
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G‐rich sequences can adopt four‐stranded helical structures, called G‐quadruplexes, that self‐assemble around monovalent cations like sodium (Na+) and potassium (K+). Whether similar structures can be formed from xeno‐nucleic acid (XNA) polymers with a shorter backbone repeat unit is an unanswered question with significant implications on the fold space of functional XNA polymers. Here, we examine the potential for TNA (α‐l ‐threofuranosyl nucleic acid) to adopt a four‐stranded helical structure based on a planar G‐quartet motif. Using native polyacrylamide gel electrophoresis (PAGE), circular dichroism (CD) and solution‐state nuclear magnetic resonance (NMR) spectroscopy, we show that despite a backbone repeat unit that is one atom shorter than the backbone repeat unit found in DNA and RNA, TNA can self‐assemble into stable G‐quadruplex structures that are similar in thermal stability to equivalent DNA structures. However, unlike DNA, TNA does not appear to discriminate between Na+ and K+ ions, as G‐quadruplex structures form equally well in the presence of either ion. Together, these findings demonstrate that despite a shorter backbone repeat unit, TNA is capable of self‐assembling into stable G‐quadruplex structures. 相似文献
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Zoetendal EG Booijink CC Klaassens ES Heilig HG Kleerebezem M Smidt H de Vos WM 《Nature protocols》2006,1(2):954-959
The human gastrointestinal (GI) tract contains a complex microbial community that consists of numerous uncultured microbes. Therefore, nucleic-acid-based approaches have been introduced to study microbial diversity and activity, and these depend on the proper isolation of DNA, rRNA and mRNA. Here, we present an RNA isolation protocol that is suitable for a wide variety of GI tract samples. The procedure for isolating DNA from GI tract samples is described in another Nature Protocols article. One of the benefits of our RNA isolation protocol is that sampling can be performed outside the laboratory, which offers possibilities for implementation in large intervention studies. The RNA isolation is based on mechanical disruption, followed by isolation of nucleic acids using phenol:chloroform:isoamylalcohol extraction and removal of DNA. In our laboratory, this protocol has resulted in the isolation of rRNA and mRNA of sufficient quality and quantity for microbial diversity and activity studies. Depending on the number of samples, the sample type and the quenching procedure chosen, the whole procedure can be performed within 2.5-4 h. 相似文献
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K Manning 《Analytical biochemistry》1991,195(1):45-50
The purification of nucleic acids from plant tissue is often made difficult by the presence of contaminating carbohydrate polymers and polyphenols. A procedure for the simultaneous isolation of DNA and translatable RNA from plants is described. The method removes most of the polysaccharides and polyphenols extracted with nucleic acids in a single step by taking advantage of differences in solubility of these compounds in the solvent 2-butoxyethanol. Stepwise addition of 2-butoxyethanol to phenol extracts of specific ionic strength precipitates nucleic acids largely free of contaminants. Subsequent separation of RNA from DNA by precipitation with LiCl was optimised to give a high recovery of translationally active RNA. Successful isolation of nucleic acids from strawberry (Fragaria X ananassa) receptacle, a particularly recalcitrant tissue, and from a range of tissues of other plant species demonstrates the general applicability of the method. 相似文献
17.
Commercial nucleic acid extraction kits are a cost effective, efficient and convenient way to isolate DNA and RNA from bacteria. Despite the increasing importance of the gastrointestinal pathogen, Clostridium difficile, and the increased use of nucleic acids in its identification, characterization, and investigation of virulence factors, no standardized or recommended methods for nucleic acid isolation exist. Here, we sought to evaluate 4 commercial DNA extraction kits and 3 commercial RNA extraction kits assessing cost, labor intensity, purity, quantity and quality of nucleic acid preparations. The DNA extraction kits produced a range of concentrations (20.9–546 ng/ml) and A260/280 ratios (1.92–2.11). All kits were suitable for DNA extraction with the exception of the Roche MagNA pure LC DNA isolation kit III which produced DNA of high yield but with substantial shearing, but that did not affect downstream PCR amplifications. For RNA extraction, the Qiagen RNeasy mini kit stood out producing preparations of consistently higher concentrations and higher RNA integrity numbers (RIN). The Roche MagNA pure LC RNA isolation kit produced preparations that could not be properly assigned RINs due to a failure to remove small RNAs which were interpreted as degradation. Good DNA and RNA yield are critical but methods are often overlooked. This study highlights the potential for critical variation between established commercial systems and the need for assessment of any extraction methods that are used. 相似文献
18.
Sira Echevarría-Zomeño Nieves Abril Julia Ruiz-Laguna Jesús Jorrín-Novo Ana M. Maldonado-Alconada 《Acta Physiologiae Plantarum》2012,34(2):793-805
Isolation of high-quality RNA and genomic DNA (gDNA) from many samples is a necessary step before accomplishing molecular
biology studies. The particular composition of Quercus ilex leaves, specially hard and rich in cell wall material, polyphenolics and secondary metabolites, usually results in preparations
contaminated with non-nucleic acid compounds. Although many methods have been developed, each case of study demands a protocol
adapted to the specific plant sample and the pursued research objectives. We have evaluated several protocols to establish
the methodology that best suited to our current genetic and molecular studies on Q. ilex. Our priority was to select the simplest methods reducing the plant starting material and the time employed, without compromising
yield, quality and integrity of the isolated nucleic acids. Our results point to two protocols based on silica-membrane purification,
as the most convenient for Q. ilex leaf tissue, and both procedures are greatly improved by adding insoluble polyvinyl polypyrrolidone during the isolation
process. The protocols optimized here can be completed at the microfuge scale and allow a researcher to process 48 samples
in 1 h, producing high quality preparations suitable for the routinely molecular biology applications with higher efficiency
than other more labour and time-consuming protocols. 相似文献
19.
Xin-wei Wang Ai-sheng Xiong Quan-hong Yao Zhen Zhang Yu-shan Qiao 《Molecular biotechnology》2010,44(1):61-65
Low molecular weight RNA (LMW RNA) is generally obtained either from the total RNA or from total nucleic acids solution. Many
steps and chemical reagents are involved in traditional methods for LMW RNA isolation where degradation of LMW RNA often occurs,
especially for plant materials with high levels of secondary catabolites. In this study, an efficient method was developed
to directly isolate pure LMW RNA from pear peel, a material rich in polyphenolics that is covered with a layer of wax. The
method was based on polyethylene glycol (PEG) precipitation combining CTAB buffer which is often used to isolate RNA from
polysaccharide-rich and polyphenolics-rich materials. The entire procedure could be completed within 6 h and many samples
could be processed at the same time. Few and common chemicals are used with this method. Hence, it could be used as an ordinary
method in the laboratory. The developed method was further tested by isolating LMW RNA from Arabidopsis. Using the isolated LMW RNA samples, microRNAs were successfully detected and characterized. 相似文献