共查询到20条相似文献,搜索用时 15 毫秒
1.
Andrea Barco Michael J. Raupach Silke Laakmann Hermann Neumann Thomas Knebelsberger 《Molecular ecology resources》2016,16(1):288-297
Sequence‐based specimen identification, known as DNA barcoding, is a common method complementing traditional morphology‐based taxonomic assignments. The fundamental resource in DNA barcoding is the availability of a taxonomically reliable sequence database to use as a reference for sequence comparisons. Here, we provide a reference library including 579 sequences of the mitochondrial cytochrome c oxidase subunit I for 113 North Sea mollusc species. We tested the efficacy of this library by simulating a sequence‐based specimen identification scenario using Best Match, Best Close Match (BCM) and All Species Barcode (ASB) criteria with three different threshold values. Each identification result was compared with our prior morphology‐based taxonomic assignments. Our simulation resulted in 87.7% congruent identifications (93.8% when excluding singletons). The highest number of congruent identifications was obtained with BCM and ASB and a 0.05 threshold. We also compared identifications with genetic clustering (Barcode Index Numbers, BINs) computed by the Barcode of Life Datasystem (BOLD). About 68% of our morphological identifications were congruent with BINs created by BOLD. Forty‐nine sequences were clustered in 16 discordant BINs, and these were divided in two classes: sequences from different species clustered in a single BIN and conspecific sequences divided in more BINs. Whereas former incongruences were probably caused by BOLD entries in need of a taxonomic update, the latter incongruences regarded taxa requiring further investigations. These include species with amphi‐Atlantic distribution, whose genetic structure should be evaluated over their entire range to produce a reliable sequence‐based identification system. 相似文献
2.
Zhang J 《Molecular ecology resources》2010,10(6):935-941
With the development of the DNA barcoding project, a large number of specimens are required to establish the library of reference barcode. Formalin-fixed samples from museums provide a potential resource for it. However, recovery of DNA and amplification of the target gene from formalin-fixed samples are challenging. In this study, a hot alkali pre-treatment accompanied by the use of cetyltrimethylammonium bromide (CTAB) method was employed for DNA recovery from formalin-preserved samples, with the purpose of pursuing the optimal condition for high quantity and quality of DNA and minimizing PCR inhibition. Meanwhile, a semi-nested PCR-based method was developed to enhance the efficacy of amplification. This advanced protocol was demonstrated to be reliable and effective. Even for 23-year-old samples, genomic DNA could be extracted, and COI gene was correctly sequenced. 相似文献
3.
DNA Barcoding (DBC) is a method for taxonomic identification of animals that is based entirely on the 5′ portion of the mitochondrial
gene, cytochrome oxidase subunit I (COI-5). It can be especially useful for identification of larval forms or incomplete specimens lacking diagnostic morphological
characters. DBC can also facilitate the discovery of species and in defining “molecular taxonomic units” in problematic groups.
However, DBC is not a panacea for coral reef taxonomy. In two of the most ecologically important groups on coral reefs, the
Anthozoa and Porifera, COI-5 sequences have diverged too little to be diagnostic for all species. Other problems for DBC include paraphyly in mitochondrial
gene trees and lack of differentiation between hybrids and their maternal ancestors. DBC also depends on the availability
of databases of COI-5 sequences, which are still in early stages of development. A global effort to barcode all fish species has demonstrated
the importance of large-scale coordination and is yielding promising results. Whether or not COI-5 by itself is sufficient for species assignments has become a contentious question; it is generally advantageous to use
sequences from multiple loci. 相似文献
4.
Sachiko Shiokai Hiroyasu Kitashiba Kenta Shirasawa Kuniaki Nagano Takeshi Nishio 《Molecular breeding : new strategies in plant improvement》2009,23(2):329-336
DNA preparation is indispensable for genotyping by DNA polymorphism analysis, and that for a large number of plants is laborious.
In the present study, a small leaf disk of rice, 1–2 mm in diameter, punched by a mini cork borer was found to be directly
usable as a PCR template. DNA fragments <300 bp were amplified efficiently. Leaf disks of 1–1.5 mm in diameter were better
than those of 2 mm for a small volume of reaction mixture. Multiplex PCR was possible with four or eight primer pairs using
the small leaf disk as a template. Leaf disks of Arabidopsis, Lotus, wheat, soybean, tomato, Chinese cabbage, and melon were also good PCR templates. This method for preparation of PCR templates,
named the leaf-punch method, was applicable to SNP analysis of a large number of plants by dot-blot-SNP analysis.
Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. 相似文献
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建立了一种简单处理单个卵子和早期胚胎制备DNA模板的方法——KOH/DTT-Triton X裂解法,并与TE-蛋白酶K法比较了PCR扩增效率。结果,采用KOH/DTT-Triton X裂解法处理单个卵子或2-细胞胚、8-细胞胚、桑椹胚、囊胚后,作为DNA模板直接进行PCR扩增线粒体DNA片段,3对引物的PCR扩增总成功率为100%(70/70),而TE-蛋白酶K法处理的单个卵子的PCR扩增总成功率为92.9%(65/70),二者差异显著(P<0.05)。但两种方法所制备模板的PCR假阳性率均为0。实验设计的KOH/DTT-Triton X裂解法是一种有效的单个早期胚胎的DNA模板制备方法,经一次PCR扩增即能获得清晰的目的DNA条带,能够满足早期胚胎遗传物质检测的需要。 相似文献
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The issue of mitochondrial heteroplasmy has been cited as a theoretical problem for DNA barcoding but is only beginning to be examined in natural systems. We sequenced multiple DNA extractions from 20 individuals of four Hawaiian Hylaeus bee species known to be heteroplasmic. All species showed strong differences at polymorphic sites between abdominal and muscle tissue in most individuals, and only two individuals had no obvious segregation. Two specimens produced completely clean sequences from abdominal DNA. The fact that these differences are clearly visible by direct sequencing indicates that substantial intra-individual mtDNA diversity may be overlooked when DNA is taken from small tissue fragments. At the same time, differences in haplotype distribution among individuals may result in incorrect recognition of cryptic species. Because DNA barcoding studies typically use only a small fragment of an organism, they are particularly vulnerable to sequencing bias where heteroplasmy and haplotype segregation are present. It is important to anticipate this possibility prior to undertaking large-scale barcoding projects to reduce the likelihood of haplotype segregation confounding the results. 相似文献
9.
Rapid DNA preparation for the quick screening is highly demanded in diverse research fields. Here, we combined an extraction buffer and heat treatment to generate DNA templates from yeast and filamentous fungal materials for PCR. This method may be widely applicable to diverse fungal species in clinical and basic studies. 相似文献
10.
Use of DNA from dry leaves for PCR and RAPD analysis 总被引:11,自引:0,他引:11
Fresh or frozen tissue is usually used as a source of DNA for PCR and RAPD analysis. We have found that leaves can be allowed
to dry at room temperature before extraction of DNA. Heating the leaves or microwave drying resulted in poor recovery of DNA.
Storage of fresh leaves in paper envelopes in the laboratory was the most successful approach. This allowed the tissue to
dry out over a period of several days and DNA could be extracted at any time, providing a convenient method for the collection
and analysis of field material. DNA from leaves stored for four months at room temperature was suitable for PCR analysis. 相似文献
11.
Eric Coissac Peter M. Hollingsworth Sébastien Lavergne Pierre Taberlet 《Molecular ecology》2016,25(7):1423-1428
DNA barcoding has had a major impact on biodiversity science. The elegant simplicity of establishing massive scale databases for a few barcode loci is continuing to change our understanding of species diversity patterns, and continues to enhance human abilities to distinguish among species. Capitalizing on the developments of next generation sequencing technologies and decreasing costs of genome sequencing, there is now the opportunity for the DNA barcoding concept to be extended to new kinds of genomic data. We illustrate the benefits and capacity to do this, and also note the constraints and barriers to overcome before it is truly scalable. We advocate a twin track approach: (i) continuation and acceleration of global efforts to build the DNA barcode reference library of life on earth using standard DNA barcodes and (ii) active development and application of extended DNA barcodes using genome skimming to augment the standard barcoding approach. 相似文献
12.
Laura Z. Rubsam Donna S. Shewach 《Journal of chromatography. B, Analytical technologies in the biomedical and life sciences》1997,702(1-2)
To isolate DNA for nucleoside analog incorporation studies, many investigators use RNase A to remove RNA from total cellular nucleic acid. We observed persistence of ribonucleotides from RNA in nucleic acid samples treated with RNase A alone. Although incubation of [5-3H]uridine-labeled nucleic acid with 50 μg/ml RNase A decreased tritium by 97%, HPLC analysis of the resulting DNA preparation digested to nucleosides revealed high levels of ribonucleosides. Increasing RNase A 10-fold (500 μg/ml) effected only a 1.7-fold reduction in ribonucleosides. Overall, the level of ribonucleosides was one-fourth that of the deoxynucleosides, primarily due to the high levels of guanosine. It was hypothesized that the ribonucleosides originated from guanosine-rich tracts of RNA since RNase A cuts preferentially 3′ to pyrimidine monophosphates and to some extent after AMP. The addition of 0.05 μg/ml RNase T1, which preferentially cleaves RNA 3′ to GMP, decreased total ribonucleosides by nearly 20-fold. In conclusion, we have developed a rapid method which removes greater then 99% of cellular RNA from nucleic acid extracts and a reversed-phase HPLC procedure that detects RNA contamination more sensitively than [5-3H]uridine labeling. These methods are useful for the determination of analog incorporation into DNA, especially for agents which incorporate into both DNA and RNA. 相似文献
13.
The chloroplast maturase K gene (matK) is one of the most variable coding genes of angiosperms and has been suggested to be a "barcode" for land plants. However, matK exhibits low amplification and sequencing rates due to low universality of currently available primers and mononucleotide repeats. To resolve these technical problems, we evaluated the entire matK region to find a region of 600-800 bp that is highly variable, represents the best of all matK regions with priming sites conservative enough to design universal primers, and avoids the mononucleotide repeats. After careful evaluation, a region in the middle was chosen and a pair of primers named natK472F and matK1248R was designed to amplify and sequence the matK fragment of approximately 776 bp. This region encompasses the most variable sites, represents the entire matK region best, and also exhibits high amplification rates and quality of sequences. The universality of this primer pair was tested using 58 species from 47 families of angiosperm plants. The primers showed a strong amplification (93.1%) and sequencing (92.6%)successes in the species tested. We propose that the new primers will solve, in part, the problems encountered when using matK and promote the adoption of matK as a DNA barcode for angiosperms. 相似文献
14.
DNA extraction from minute hymenopterans and their larvae is difficult and challenging because of their small size indicating a low amount of starting material. Hence, 11 DNA extraction methods were compared to determine their efficacy in isolating DNA. Success of each method was scored on a 2% agarose gel after PCR of the cox 1 mitochondrial locus. A silica-membrane-based approach was the most successful, followed by a method using a combination of incubation buffers and a method using magnetic beads. The method using buffers was the most cost- and time effective. Using this method, larvae from Eucalyptus seed capsule galls could be assigned a role (parasitoid, gall former or inquiline) in the gall-inhabiting complex. 相似文献
15.
The second internal transcribed spacer (ITS2) of the nuclear ribosomal RNA cluster (rDNA) is significantly smaller in the Cnidaria (120–260 bp) than in the rest of the Metazoa. ITS2 is one of the fastest evolving DNA regions among those commonly used in molecular systematics and has been proposed as a possible barcoding gene for Cnidaria to replace the currently problematic mitochondrial sequences used. We have reviewed the intraspecific and interspecific variation of ITS2 rRNA sequences in the Anthozoa. We have observed that the lower limits of the interspecific DNA divergence ranges very often overlap with intraspecific ranges, and identical sequences from individuals of different species are not rare. This finding can result in problems similar to those encountered with the mitochondrial COI, and we conclude that ITS2 does not prove significantly better than COI for standard taxonomic DNA barcoding in Anthozoa. However, ITS2 appears to be a promising gene in the ecological DNA barcoding of corallivory, where taxonomic accuracy at genus or even family level may represent a significant improvement of current knowledge. We have successfully amplified and sequenced ITS2 from template DNA extracted from foot muscle and from stomach contents of corallivorous gastropods, and from their anthozoan hosts. The small size of cnidarian ITS2 makes it a very easy and efficient tool for ecological barcoding of associations. Ecological barcoding of corallivory is an indispensable approach to the study of the associations in deep water, where direct observation is severely limited by logistics and costs. 相似文献
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Alex D. Twyford 《Molecular ecology》2014,23(19):4674-4676
The goal of DNA barcoding is to enable the rapid identification of taxa from short diagnostic DNA sequence profiles. But how feasible is this objective when many evolutionary processes, such as hybridization and selective sweeps, cause alleles to be shared among related taxa? In this issue of Molecular Ecology, Percy et al. (2014) test the full suite of seven candidate plant barcoding loci in a broad geographic sample of willow species. They show exceptional plastid haplotype sharing between species across continents, with most taxa not possessing a unique barcode sequence. Using population genetic and molecular dating analyses, they implicate hybridization and selective sweeps, but not incomplete lineage sorting, as the historical processes causing widespread haplotype sharing among willow taxa. This study represents an exceptional case of how poorly barcoding can perform, and highlights methodological issues using universal organellar regions for species identification. 相似文献
19.
For isolation of fungal DNA for PCR amplification, we compared three DNA isolation methods: enzymatic cleavage and the use of benzyl chloride or benzyl bromide. Since benzyl bromide is more reactive, its use enabled us to readily isolate the total nucleic acids as a DNA template source from various fungi, including dematiaceous hyphomycetes, for RAPD analysis. 相似文献
20.
Three PCR-based methods are described that allow covalent drug-DNA adducts, and their repair, to be studied at various levels
of resolution from gene regions to the individual nucleotide level in single copy genes. A quantitative PCR (QPCR) method
measures the total damage on both DNA strands in a gene region, usually between 300 and 3000 base pairs in length. Strand-specific
QPCR incorporates adaptations that allow damage to be measured in the same region as QPCR but in a strand-specific manner.
Single-strand ligation PCR allows the detection of adduct formation at the level of single nucleotides, on individual strands,
in a single copy gene in mammalian cells. If antibodies to the DNA adducts of interest are available, these can be used to
capture and isolate adducted DNA for use in single-strand ligation PCR increasing the sensitivity of the assay. 相似文献