共查询到20条相似文献,搜索用时 31 毫秒
1.
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. 相似文献
2.
We have used the polymerase chain reaction (PCR) method to monitor meiotic recombination in the basidiomycete Coprinus cinereus. We used DNA-mediated transformation to recover strains with modifications of the trp1 locus. The modifications were designed to introduce unique PCR priming sites separated by a homologous 2.4?kb region in which crossing over could occur. We showed that exchange occurred in this region at the frequency expected for a typical region of this genome (2.4?kb should correspond to a genetic length of 0.08?cM). We also detected products resulting from crossing over in DNAs extracted from cells in meiotic prophase. The assay should be useful for monitoring exchange in mutants that cannot complete meiosis. 相似文献
3.
Estimates of past forest composition obtained from Late Quaternary pollen spectra via a calibration of modern pollen spectra in terms of species abundances are subject to various sources of error, whose combined effect requires statistical analysis. Two statistical procedures, the maximum likelihood method and an approach using series expansions, are used to estimate standard deviations associated with forest composition estimates obtained via the R-value method of calibration. The two approaches yield similar values. The series expansion method also allows one to allocate pollen counting effort between fossil and modern samples in such a way as to maximize the precision of the final estimates. M.B. Davis's original controversial estimates of early Holocene forest composition in Vermont, U.S.A., are shown to have been vitiated by statistical errors. The optimum allocation procedure here suggests increasing the relative effort put into the modern count. This change would have improved but not rescued the estimates; omission of Larix, however, led to a substantial reduction in the errors. Exceptionally poor pollen producers such as Larix should generally be excluded from quantitative calibration; the remaining taxa should be calibrated on the basis of large samples of pollen, the modern pollen being collected preferably from a network of surface sampling sites. 相似文献
4.
Distribution of Gene Frequency as a Test of the Theory of the Selective Neutrality of Polymorphisms 总被引:44,自引:10,他引:34
下载免费PDF全文
![点击此处可从《Genetics》网站下载免费的PDF全文](/ch/ext_images/free.gif)
The variation in gene frequency among populations or between generations within a population is a result of breeding structure and selection. But breeding structure should affect all loci and alleles in the same way. If there is significant heterogeneity between loci in their apparent inbreeding coefficients F=sp2/p(1-p), this heterogeneity may be taken as evidence for selection. We have given the statistical properties of F and shown how tests of heterogeneity can be made. Using data from human populations we have shown highly significant heterogeneity in F values for human polymorphic genes over the world, thus demonstrating that a significant fraction of human polymorphisms owe their current gene frequencies to the action of natural selection. We have also applied the method to temporal variation within a population for data on Dacus oleae and have found no significant evidence of selection. 相似文献
5.
Rapid high resolution single nucleotide polymorphism-comparative genome hybridization mapping in Caenorhabditis elegans
下载免费PDF全文
![点击此处可从《Genetics》网站下载免费的PDF全文](/ch/ext_images/free.gif)
We have developed a significantly improved and simplified method for high-resolution mapping of phenotypic traits in Caenorhabditis elegans using a combination of single nucleotide polymorphisms (SNPs) and oligo array comparative genome hybridization (array CGH). We designed a custom oligonucleotide array using a subset of confirmed SNPs between the canonical wild-type Bristol strain N2 and the Hawaiian isolate CB4856, populated with densely overlapping 50-mer probes corresponding to both N2 and CB4856 SNP sequences. Using this method a mutation can be mapped to a resolution of ~200 kb in a single genetic cross. Six mutations representing each of the C. elegans chromosomes were detected unambiguously and at high resolution using genomic DNA from populations derived from as few as 100 homozygous mutant segregants of mutant N2/CB4856 heterozygotes. Our method completely dispenses with the PCR, restriction digest, and gel analysis of standard SNP mapping and should be easy to extend to any organism with interbreeding strains. This method will be particularly powerful when applied to difficult or hard-to-map low-penetrance phenotypes. It should also be possible to map polygenic traits using this method. 相似文献
6.
7.
Prediction of signal recognition particle RNA genes 总被引:3,自引:1,他引:3
We describe a method for prediction of genes that encode the RNA component of the signal recognition particle (SRP). A heuristic search for the strongly conserved helix 8 motif of SRP RNA is combined with covariance models that are based on previously known SRP RNA sequences. By screening available genomic sequences we have identified a large number of novel SRP RNA genes and we can account for at least one gene in every genome that has been completely sequenced. Novel bacterial RNAs include that of Thermotoga maritima, which, unlike all other non-gram-positive eubacteria, is predicted to have an Alu domain. We have also found the RNAs of Lactococcus lactis and Staphylococcus to have an unusual UGAC tetraloop in helix 8 instead of the normal GNRA sequence. An investigation of yeast RNAs reveals conserved sequence elements of the Alu domain that aid in the analysis of these RNAs. Analysis of the human genome reveals only two likely genes, both on chromosome 14. Our method for SRP RNA gene prediction is the first convenient tool for this task and should be useful in genome annotation. 相似文献
8.
Janine T. Bossé Denise M. Soares-Bazzolli Yanwen Li Brendan W. Wren Alexander W. Tucker Duncan J. Maskell Andrew N. Rycroft Paul R. Langford 《PloS one》2014,9(11)
We have developed a simple method of generating scarless, unmarked mutations in Actinobacillus pleuropneumoniae by exploiting the ability of this bacterium to undergo natural transformation, and with no need to introduce plasmids encoding recombinases or resolvases. This method involves two successive rounds of natural transformation using linear DNA: the first introduces a cassette carrying cat (which allows selection by chloramphenicol) and sacB (which allows counter-selection using sucrose) flanked by sequences to either side of the target gene; the second transformation utilises the flanking sequences ligated directly to each other in order to remove the cat-sacB cassette. In order to ensure efficient uptake of the target DNA during transformation, A. pleuropneumoniae uptake sequences are added into the constructs used in both rounds of transformation. This method can be used to generate multiple successive deletions and can also be used to introduce targeted point mutations or insertions of heterologous genes into the A. pleuropneumoniae chromosome for development of live attenuated vaccine strains. So far, we have applied this method to highly transformable isolates of serovars 8 (MIDG2331), which is the most prevalent in the UK, and 15 (HS143). By screening clinical isolates of other serovars, it should be possible to identify other amenable strains. 相似文献
9.
Cristina Gruas Isidro Álvarez Carlos Lara Cristina Belén García Demetris Savva M. Victoria Arruga 《Indian journal of microbiology》2013,53(2):142-148
Rapid and more sensitive methods for the detection and quantification of viable Legionella cells have been developed. In this paper, a comparative analysis of environmental water samples using the ScanVIT-Legionella? method and the traditional “gold standard” method of culturing is realised indicating the usefulness of the ScanVIT method. The ScanVIT-Legionella? method was performed on environmental water samples from different locations of Huesca region (Spain). Legionella micro-colonies should appear green colour and Legionella pneumophila micro-colonies appear red. Twenty-one environmental water samples analysed by standard culture plus five control samples (Two sterile water samples with Legionella as positive controls and three sterile water samples as negative controls). All of them were used to apply ScanVIT-Legionella? method. From of 21 environmental samples eleven were positive, six negative with both methods and four samples were negative for culture method and positive for ScanVIT-Legionella? method. The positive control samples were positive and the negative were negative for both methods. A comparative analysis of the results obtained with two methods showed a strong positive determination coefficient (R2 = 0.99753). The results demonstrate the usefulness of the ScanVIT-Legionella? method as a rapid diagnostic tool in order to provide a diagnosis as quick as possible. ScanVIT-Legionella? method offers a series of advantages such as quickly diagnosis, higher sensitivity and the possibility to identify Legionella spp. and L. pneumophila simultaneously. 相似文献
10.
Thomas Emil Andersen Peter Kingshott Maike Benter Hans Jørn Kolmos 《Journal of microbiological methods》2010,81(2):135-140
We have established a simple flow chamber-based procedure which provides an accurate and reproducible way to measure the amount of biofilm formed on an implantable biomaterial surface. The method enables the side-by-side evaluation of different materials under hydrodynamic flow conditions similar to those found on an implanted device. We have used the method to evaluate the biofilm forming capacity of clinically isolated Escherichia coli on silicone rubber and on silicone rubber containing a hydrophilic coating. It was found that the surface chemistry influenced the colonization of the isolates very differently. In addition, the temperature was found to have a considerable influence upon the adhesion and biofilm forming capacity of some of the isolates, and that the influence of surface chemistry depended on temperature. Our results suggest that the step from using E. coli laboratory strains to clinical isolates entails a significant rise in complexity and yields results that cannot be generalized. The results should be valuable information for researchers working with pre-clinical evaluation of device-associated E. coli infections. 相似文献
11.
Jeffry D. Sander Cherie L. Ramirez Samantha J. Linder Vikram Pattanayak Noam Shoresh Manching Ku Jennifer A. Foden Deepak Reyon Bradley E. Bernstein David R. Liu J. Keith Joung 《Nucleic acids research》2013,41(19):e181
Gene-editing nucleases enable targeted modification of DNA sequences in living cells, thereby facilitating efficient knockout and precise editing of endogenous loci. Engineered nucleases also have the potential to introduce mutations at off-target sites of action. Such unintended alterations can confound interpretation of experiments and can have implications for development of therapeutic applications. Recently, two improved methods for identifying the off-target effects of zinc finger nucleases (ZFNs) were described–one using an in vitro cleavage site selection method and the other exploiting the insertion of integration-defective lentiviruses into nuclease-induced double-stranded DNA breaks. However, application of these two methods to a ZFN pair targeted to the human CCR5 gene led to identification of largely non-overlapping off-target sites, raising the possibility that additional off-target sites might exist. Here, we show that in silico abstraction of ZFN cleavage profiles obtained from in vitro cleavage site selections can greatly enhance the ability to identify potential off-target sites in human cells. Our improved method should enable more comprehensive profiling of ZFN specificities. 相似文献
12.
13.
《Harmful algae》2012
Microscopy, a staple of monitoring programs for tracking the occurrence and abundance of harmful algal bloom (HAB) species, is time consuming and often characterized by high uncertainty. Alternate methods that allow rapid and accurate assessment of presence and abundance of HAB species are needed. For many HAB species, such as the toxigenic haptophyte, Prymnesium parvum, molecular methods including quantitative real-time PCR (qPCR) have been developed with the suggestion that they should be useful for monitoring programs. However, this suggestion rarely has been put into action. In this study, we modified a recently developed method for detecting P. parvum using qPCR and tested its efficacy as an alternative to microscopy for P. parvum detection and enumeration in a long-term monitoring program in a recently invaded subtropical US reservoir. Abundance estimates of P. parvum were similar for both methods, but we detected P. parvum at multiple sites using qPCR where it previously had gone undetected by microscopy. Using qPCR, we substantially reduced processing time, increased detection limit and reduced error in P. parvum abundance estimates compared to microscopy. Thus, qPCR is an effective tool for detecting and monitoring P. parvum, particularly at pre-bloom densities, and should likewise prove useful in monitoring programs for the other HAB species for which qPCR methods have been developed. 相似文献
14.
Richard S. Shapiro Ben C. Eisenberg William Binder 《The Western journal of medicine》1965,103(5):340-342
A two-year survey of pollen and mold spores by the gravity slide method revealed that there are no clear-cut tree, grass or weed pollen seasons in California.Pollen counts should be correlated with field studies to distinguish the various plants whose pollen have a similar appearance.Spores of Alternaria and Hormodendrum, whose importance in allergic disease of the respiratory tract has been well established for many years, were found all during the year. More hormodendrum spores were collected than the total of all other pollens combined. 相似文献
15.
New Unstable Variants of Green Fluorescent Protein for Studies of Transient Gene Expression in Bacteria 总被引:36,自引:8,他引:28
下载免费PDF全文
![点击此处可从《Applied microbiology》网站下载免费的PDF全文](/ch/ext_images/free.gif)
Jens Bo Andersen Claus Sternberg Lars Kongsbak Poulsen Sara Petersen Bjrn Michael Givskov Sren Molin 《Applied microbiology》1998,64(6):2240-2246
Use of the green fluorescent protein (Gfp) from the jellyfish Aequorea victoria is a powerful method for nondestructive in situ monitoring, since expression of green fluorescence does not require any substrate addition. To expand the use of Gfp as a reporter protein, new variants have been constructed by the addition of short peptide sequences to the C-terminal end of intact Gfp. This rendered the Gfp susceptible to the action of indigenous housekeeping proteases, resulting in protein variants with half-lives ranging from 40 min to a few hours when synthesized in Escherichia coli and Pseudomonas putida. The new Gfp variants should be useful for in situ studies of temporal gene expression. 相似文献
16.
Dressman HK Barley-Maloney L Rowlette LL Agris PF Garcia-Blanco MA 《Nucleic acids research》2006,34(19):e131
En masse analysis of gene structure and function by array technologies will have a lasting and profound effect on biology and medicine. This impact can be compromised by low quality of probes within arrays, which we show can be caused by incomplete removal of chemical protecting groups. To solve this quality control problem, we present a sensitive, specific and facile method to detect these groups in situ on arrays using monoclonal antibodies and existing instrumentation. Screening of microarrays with these monoclonal antibodies should guide the consideration given to data derived from these and should enhance the accuracy of the results obtained. 相似文献
17.
Change point detectors (CPDs) are used to segment recordings of single molecules for the purpose of kinetic analysis. The assessment of the accuracy of CPD algorithms has usually been based on testing them with simulated data. However, there have not been methods to assess the output of CPDs from real data independent of simulation. Here we present one method to do this based on the assumption that the elementary kinetic unit is a stationary period (SP) with a normal distribution of samples, separated from other SPs by change points (CPs). Statistical metrics of normality can then be used to assess SPs detected by a CPD algorithm (detected SPs, DSPs). Two statistics in particular were found to be useful, the z-transformed skew (S Z) and z-transformed kurtosis (K Z). K Z(S Z) plots of DSP from noise, simulated data and single ion channel recordings showed that DSPs with false negative CP could be distinguished. Also they showed that filtering had a significant effect on the normality of data and so filtering should be taken into account when calculating statistics. This method should be useful for analyzing single molecule recordings where there is no simple model for the data. 相似文献
18.
A modification of the benzoylated DEAE-cellulose method of tRNA fractionation has been developed to provide a rapid and highly efficient method for the quantitative identification of multiple tryptophan, tyrosine, and phenylalanine tRNA species from E. coli aminoacylated in vivo. This method should be of particular use in physiological studies of these tRNA species. 相似文献
19.
20.
Discontinuous DNA replication: accumulation of Simian virus 40 DNA at specific stages in its replication. 总被引:17,自引:0,他引:17
The number of proline residues in a protein should have very marked consequences for the rates of protein unfolding and refolding according to the model proposed by Brandts et al. (1975). Kinetic simulations of this model indicate that the half-time for refolding of a polypeptide chain with 20 proline residues should be greater than 10 minutes and should increase by about an order of magnitude for each additional 10 proline residues. Various means are considered by which the rate of protein folding in vivo and in vitro might be increased. 相似文献