In vivo generation of DNA sequence diversity for cellular barcoding

Heterogeneity is a ubiquitous feature of biological systems. A complete understanding of such systems requires a method for uniquely identifying and tracking individual components and their interactions with each other. We have developed a novel method of uniquely tagging individual cells in vivo with a genetic ‘barcode’ that can be recovered by DNA sequencing. Our method is a two-component system comprised of a genetic barcode cassette whose fragments are shuffled by Rci, a site-specific DNA invertase. The system is highly scalable, with the potential to generate theoretical diversities in the billions. We demonstrate the feasibility of this technique in Escherichia coli. Currently, this method could be employed to track the dynamics of populations of microbes through various bottlenecks. Advances of this method should prove useful in tracking interactions of cells within a network, and/or heterogeneity within complex biological samples.     

The effect of geographical scale of sampling on DNA barcoding

Eight years after DNA barcoding was formally proposed on a large scale, CO1 sequences are rapidly accumulating from around the world. While studies to date have mostly targeted local or regional species assemblages, the recent launch of the global iBOL project (International Barcode of Life), highlights the need to understand the effects of geographical scale on Barcoding's goals. Sampling has been central in the debate on DNA Barcoding, but the effect of the geographical scale of sampling has not yet been thoroughly and explicitly tested with empirical data. Here, we present a CO1 data set of aquatic predaceous diving beetles of the tribe Agabini, sampled throughout Europe, and use it to investigate how the geographic scale of sampling affects 1) the estimated intraspecific variation of species, 2) the genetic distance to the most closely related heterospecific, 3) the ratio of intraspecific and interspecific variation, 4) the frequency of taxonomically recognized species found to be monophyletic, and 5) query identification performance based on 6 different species assignment methods. Intraspecific variation was significantly correlated with the geographical scale of sampling (R-square = 0.7), and more than half of the species with 10 or more sampled individuals (N = 29) showed higher intraspecific variation than 1% sequence divergence. In contrast, the distance to the closest heterospecific showed a significant decrease with increasing geographical scale of sampling. The average genetic distance dropped from > 7% for samples within 1 km, to < 3.5% for samples up to > 6000 km apart. Over a third of the species were not monophyletic, and the proportion increased through locally, nationally, regionally, and continentally restricted subsets of the data. The success of identifying queries decreased with increasing spatial scale of sampling; liberal methods declined from 100% to around 90%, whereas strict methods dropped to below 50% at continental scales. The proportion of query identifications considered uncertain (more than one species < 1% distance from query) escalated from zero at local, to 50% at continental scale. Finally, by resampling the most widely sampled species we show that even if samples are collected to maximize the geographical coverage, up to 70 individuals are required to sample 95% of intraspecific variation. The results show that the geographical scale of sampling has a critical impact on the global application of DNA barcoding. Scale-effects result from the relative importance of different processes determining the composition of regional species assemblages (dispersal and ecological assembly) and global clades (demography, speciation, and extinction). The incorporation of geographical information, where available, will be required to obtain identification rates at global scales equivalent to those in regional barcoding studies. Our result hence provides an impetus for both smarter barcoding tools and sprouting national barcoding initiatives-smaller geographical scales deliver higher accuracy.     

DNA barcoding of lichenized fungi demonstrates high identification success in a floristic context

? Efforts are currently underway to establish a standard DNA barcode region for fungi; we tested the utility of the internal transcribed spacer (ITS) of nuclear ribosomal DNA for DNA barcoding in lichen-forming fungi by sampling diverse species across eight orders. ? Amplification of the ITS region (ITS1-5.8S-ITS2) was conducted for 351 samples, encompassing 107, 55 and 28 species, genera and families, respectively, of lichenized fungi. We assessed the ability of the entire ITS vs the ITS2 alone to discriminate between species in a taxonomic dataset (members of the genus Usnea) and a floristic dataset. ? In the floristic dataset, 96.3% of sequenced samples could be assigned to the correct species using ITS or ITS2; a barcode gap for ITS is present in 92.1% of species. Although fewer species have a barcode gap in the taxonomic dataset (73.3% with ITS and 68.8% with ITS2), up to 94.1% of samples were assigned to the correct species using BLAST. ? While discrimination between the most closely related species will remain challenging, our results demonstrate the potential to identify a high percentage of specimens to the correct species, and the remainder to the correct genus, when using DNA barcoding in a floristic context.     

DNA barcoding of billfishes

DNA barcoding is a method promising fast and accurate identification of animal species based on the sequencing of the mitochondrial c oxidase subunit (COI) gene. In this study, we explore the prospects for DNA barcoding in one particular fish group, the billfishes (suborder Xiphioidei--swordfish, marlins, spearfishes, and sailfish). We sequenced the mitochondrial COI gene from 296 individuals from the 10 currently recognized species of billfishes, and combined these data with a further 57 sequences from previously published projects. We also sequenced the rhodopsin gene from a subset of 72 individuals to allow comparison of mitochondrial results against a nuclear marker. Five of the 10 species are readily distinguishable by COI barcodes. Of the rest, the striped marlin (Kajikia audax) and white marlin (K. albida) show highly similar sequences and are not unambiguously distinguishable by barcodes alone, likewise are the three spearfishes Tetrapturus angustirostris, T. belone, and T. pfluegeri. We discuss the taxonomic status of these species groups in light of our and other data, molecular and morphological.     

DNA barcoding of blastocystis

We have developed a simple method for subtyping the intestinal protistan parasite Blastocystis using an approach equivalent to DNA barcoding in animals. Amplification of a 600 bp region of the small subunit ribosomal RNA gene followed by single primer sequencing of the PCR product provides enough data to assign isolates to specific subtypes unambiguously. We believe that this approach will prove useful in future epidemiological studies.     

O-Alkyl dioleoylphosphatidylcholinium compounds: the effect of varying alkyl chain length on their physical properties and in vitro DNA transfection activity

1,2-Dioleoyl-sn-3-ethylphosphocholine (EDOPC) has been previously shown be a highly effective DNA transfection reagent in vitro. To assess the effect of alkyl chain length on transfection efficiency, the O-methyl, O-propyl, O-hexyl, O-decyl, and O-octadecyl derivatives have been prepared from dioleoylphosphatidylcholine using the corresponding alkyl trifluoromethylsulfonate. The methyl, ethyl, and propyl derivatives formed liposomes which were very large and unilamellar. The ethyl and propyl derivatives were equally efficient at mediating transfection (even in the presence of serum) of BHK cells, but the chemically labile methyl derivative was a much weaker transfection agent. The O-decyl and O-octadecyl compounds, which assume the inverted hexagonal phase in excess water (as determined by X-ray diffraction), were almost inactive after manual agitation in both water and in saline; however, after sonication, these compounds exhibited good transfection activity. The O-hexyl derivative displayed novel behavior, assuming the lamellar phase at low and a cubic phase at high ionic strength. All compounds, whether lamellar or not, formed lamellar structures when complexed with DNA. In water, where the hexyl compound dispersed well, sonication diminished transfection activity, whereas at physiological ionic strength, which led to poor manual dispersion, sonication was essential for good transfection. These results emphasize the importance of optimal dispersion of a cationic lipid: too little, and interaction with DNA is handicapped, too much, and the resultant particle transfects poorly. Lipid dispersibility is thus an important variable in assessing lipid transfection agents, and caution is advised in attributing too much significance to chemical structure until interaction with DNA has been optimized.     

DNA barcoding of Canada's skates

DNA-based identifications have been employed across broad taxonomic ranges and provide an especially useful tool in cases where external identification may be problematic. This study explored the utility of DNA barcoding in resolving skate species found in Atlantic Canadian waters. Most species were clearly resolved, expanding the utility for such identification on a taxonomically problematic group. Notably, one genus (Amblyraja) contained three of four species whose distributions do not overlap that could not be readily identified with this method. On the other hand, two common and partially sympatric species (Little and Winter skates) were readily identifiable. There were several instances of inconsistency between the voucher identification and the DNA sequence data. In some cases, these were at the intrageneric level among species acknowledged to be prone to misidentification. However, several instances of intergeneric discrepancies were also identified, suggesting either evidence of past introgressive hybridization or misidentification of vouchered specimens across broader taxonomic ranges. Such occurrences highlight the importance of retaining vouchered specimens for subsequent re-examination in the light of conflicting DNA evidence.     

DNA barcoding of Dutch birds

The mitochondrial cytochrome c oxidase subunit I (COI) can serve as a fast and accurate marker for the identification of animal species, and has been applied in a number of studies on birds. We here sequenced the COI gene for 387 individuals of 147 species of birds from the Netherlands, with 83 species being represented by > 2 sequences. The Netherlands occupies a small geographic area and 95% of all samples were collected within a 50 km radius from one another. The intraspecific divergences averaged 0.29% among this assemblage, but most values were lower; the interspecific divergences averaged 9.54%. In all, 95% of species were represented by a unique barcode, with 6 species of gulls and skua (Larus and Stercorarius) having at least one shared barcode. This is best explained by these species representing recent radiations with ongoing hybridization. In contrast, one species, the Lesser Whitethroat Sylvia curruca showed deep divergences, averaging 5.76% and up to 8.68% between individuals. These possibly represent two distinct taxa, S. curruca and S. blythi, both clearly separated in a haplotype network analysis. Our study adds to a growing body of DNA barcodes that have become available for birds, and shows that a DNA barcoding approach enables to identify known Dutch bird species with a very high resolution. In addition some species were flagged up for further detailed taxonomic investigation, illustrating that even in ornithologically well-known areas such as the Netherlands, more is to be learned about the birds that are present.     

DNA序列分析用于常见致病真菌鉴定和分型

随着真菌感染的增多,仅用表型方法鉴定环境中或临床上的致病真菌不足以快速准确地诊断真菌感染疾病,近年来,分子生物学方法因快速、准确而逐步得到应用,其中DNA序列分析已成为鉴定致病真菌到种水平的重要方法。现就DNA序列分析在常见致病真菌分类鉴定及基因分型的应用加以综述。     

Assessing the pathogenic effect ofFusarium, Geosmithia andOphiostoma fungi from broad-leaved trees

Phytopathogenic effect of Geosmithia pallida, G. langdonii, Ophiostoma grandicarpum, O. querci, two isolates of O. piceae, and two isolates of Fusarium solani was compared using plant growth test (stem and root length of garden cress plants seeded on mycelium-covered potato carrot agar); Ophiostoma spp. and F. solani were isolated from oak, Geosmithia spp. from galleries of Scolytus intricatus on beech. All fungi inhibited more the root elongation than that of stems. F. solani led to plant collapse after briefly stimulating the growth of stem and in one case also root. G. langdonii inhibited stem and root growth to 20% and led to plant collapse. G. pallida inhibited root growth to 25% whereas stem growth was almost unimpaired. Ophiostoma spp. reduced stem growth to approximately 60-80% and root growth to 25-60%. O. piceae and O. querci caused plant collapse after 15-20 d.     

VISTA : visualizing global DNA sequence alignments of arbitrary length

Summary: VISTA is a program for visualizing global DNA sequence alignments of arbitrary length. It has a clean output, allowing for easy identification of similarity, and is easily configurable, enabling the visualization of alignments of various lengths at different levels of resolution. It is currently available on the web, thus allowing for easy access by all researchers. Availability: VISTA server is available on the web at http://www-gsd.lbl.gov/vista. The source code is available upon request. Contact: vista@lbl.gov     

Assessing the use of the mitochondrial cox1 marker for use in DNA barcoding of red algae (Rhodophyta)

The red algae, a remarkably diverse group of organisms, are difficult to identify using morphology alone. Following the proposal to use the mitochondrial cytochrome c oxidase subunit I (cox1) for DNA barcoding animals, we assessed the use of this gene in the identification of red algae using 48 samples plus 31 sequences obtained from GenBank. The data set spanned six orders of red algae: the Bangiales, Ceramiales, Corallinales, Gigartinales, Gracilariales and Rhodymeniales. The results indicated that species could be discriminated. Intraspecific variation was between 0 and 4 bp over 539 bp analyzed except in Mastocarpus stellatus (0-14 bp) and Gracilaria gracilis (0-11 bp). Cryptic diversity was found in Bangia fuscopurpurea, Corallina officinalis, G. gracilis, M. stellatus, Porphyra leucosticta and P. umbilicalis. Interspecific variation across all taxa was between 28 and 148 bp, except for G. gracilis and M. stellatus. A comparison of cox1 with the plastid Rubisco spacer for Porphyra species revealed that it was a more sensitive marker in revealing incipient speciation and cryptic diversity. The cox1 gene has the potential to be used for DNA barcoding of red algae, although a good taxonomic foundation coupled with extensive sampling of taxa is essential for the development of an effective identification system.     

The effect of sequence heterogeneity on DNA melting kinetics

We consider kinetics of the cooperative melting of DNA sections situated at the edge of the helix. Accurate calculations based on the real sequences of such sections demonstrate that their internal heterogeneity has a drastic effect on the melting kinetics. Allowance for the internal heterogeneity increases the relaxation time by several orders of magnitude as compared with a model based on the assumption of equal base-pair stability within a section. The relaxation times obtained are in good agreement with the experimental data of Suyama and Wada (A. Suyama and A. Wada, Biopolymers, 23, 409 (1984)). An analysis of the melting process revealed some simple sequence characteristics that determine its rate. An examination of the temperature dependence of the relaxation time led to a distinct interpretation of the apparent activation energies of the denaturation and renaturation. The relaxation time proved to reach its maximum near the equilibrium melting point of the section examined.     

Influence of sequence context and length on the structure and stability of triplet repeat DNA oligomers

Genetic expansion diseases have been linked to the properties of triplet repeat DNA sequences during replication. The most common triplet repeats associated with such diseases are CAG, CCG, CGG, and CTG. It has been suggested that gene expansion occurs as a result of hairpin formation of long stretches of these sequences on the leading daughter strand synthesized during DNA replication [Gellibolian, R., Bacolla, A., and Wells, R. D. (1997) J. Biol. Chem. 272, 16793-7]. To test the biophysical basis for this model, oligonucleotides of general sequence (CNG)(n), where N = A, C, G, or T and n = 4, 5, 10, 15, or 25, were synthesized and characterized by circular dichroism (CD) spectropolarimetry, optical melting studies, and differential scanning calorimetry (DSC). The goal of these studies was to evaluate the influence of sequence context and oligomer length on their secondary structures and stabilities. The results indicate that all single oligomers, even those as short as 12 nucleotides, form stable hairpin structures at 25 degrees C. Such hairpins are characterized by the presence of N:N mismatched base pairs sandwiched between G:C base pairs in the stems and loops of three to four unpaired bases. Thermodynamic analysis of these structures reveals that their stabilities are influenced by both the sequence of the particular oligomer and its length. Specifically, the stability order of CGG > CTG > CAG > CCG was observed. In addition, longer oligomers were found to be more stable than shorter oligomers of the same sequence. However, a stability plateau above 45 nucleotides suggests that the length dependence reaches a maximum value where the stability of the G:C base pairs can no longer compensate the instability of the N:N mismatches in the stems of the hairpins. The results are discussed in terms of the above model proposed for gene expansion.     

Nucleosome reconstitution: effect of DNA length on nuclesome structure.

Core histones (H2A, H2B, H3, and H4) are reconstituted by salt gradient dialysis with DNA molecules ranging in length from 177 bp down to 50 bp. While reconstituted particles containing 125 bp are very similar to native particles, those particles containing a single piece of shorter DNA tend to aggregate. The aggregation depends on the ionic strength and DNA length. The DNA placement on the histone core is not random as determined by pancreatic DNase I digestions of particles containing 32P 5'-end-labeled DNA. Rather, it is found that all DNA molecules, up to 161 bp in length, reassociate with core histones in such a way as to produce defined patterns of DNase I cutting with respect to the 5' ends. Particles were made that contained two pieces of 65-bp DNA. These particles are very similar to native particles under most conditions but tended to dissociation results in the production of two half-nucleosomes (hemisones).     

DNA barcoding of endangered Indian Paphiopedilum species

The indiscriminate collections of Paphiopedilum species from the wild for their exotic ornamental flowers have rendered these plants endangered. Although the trade of these endangered species from the wild is strictly forbidden, it continues unabated in one or other forms that elude the current identification methods. DNA barcoding that offers identification of a species even if only a small fragment of the organism at any stage of development is available could be of great utility in scrutinizing the illegal trade of both endangered plant and animal species. Therefore, this study was undertaken to develop DNA barcodes of Indian species of Paphiopedilum along with their three natural hybrids using loci from both the chloroplast and nuclear genomes. The five loci tested for their potential as effective barcodes were RNA polymerase-β subunit (rpoB), RNA polymerase-β' subunit (rpoC1), Rubisco large subunit (rbcL) and maturase K (matK) from the chloroplast genome and nuclear ribosomal internal transcribed spacer (nrITS) from the nuclear genome. The intra- and inter-specific divergence values and species discrimination rates were calculated by Kimura 2 parameter (K2P) method using mega 4.0. The matK with 0.9% average inter-specific divergence value yielded 100% species resolution, thus could distinguish all the eight species of Paphiopedilum unequivocally. The species identification capability of these sequences was further confirmed as each of the matK sequences was found to be unique for the species when a blast analysis of these sequences was carried out on NCBI. nrITS, although had 4.4% average inter-specific divergence value, afforded only 50% species resolution. DNA barcodes of the three hybrids also reflected their parentage.