Detection of genome specific monomorphic loci in Bos taurus and Bubalus bubalis with oligodeoxyribonucleotide probe

A synthetic oligodeoxyribonucleotide probe (OAT36) comprising nine repeats of 5'GACA 3′ and several enzymes were used to analyse cow, (Bos taurus) and buffalo (Bubalus bubalis) genomes and a number of monomorphic loci were detected in both the species. Different animals from the same species showed an almost ‘similar’ monomorphic hybridization pattern but animals from two separate species showed a different ‘genome specific’ pattern. The overall hybridization with any enzyme and probe combination was found to be unique to one species. This forms the basis of genome specific hybridization which is substantiated by our zoo-blot hybridization studies. The evolutionary aspect of these loci in the context of sequence polymorphisms is discussed.     

Application of fluorescence melting curve analysis for dual DNA detection using single peptide nucleic acid probe

Peptide nucleic acid (PNA) is an artificially synthesized polymer. PNA oligomers show greater specificity in binding to complementary DNAs. Using this PNA, fluorescence melting curve analysis (FMCA) for dual detection was established. Genomic DNA of Mycoplasma fermentans and Mycoplasma hyorhinis was used as a template DNA model. By using one PNA probe, M. fermentans and M. hyorhinis could be detected and distinguished simultaneously in a single tube. The developed PNA probe is a dual‐labeled probe with fluorescence and quencher dye. The PNA probe perfectly matches the M. fermentans 16s rRNA gene, with a melting temperature of 72°C. On the other hand, the developed PNA probe resulted in a mismatch with the 16s rRNA gene of M. hyorhinis, with a melting temperature of 44–45°C. The melting temperature of M. hyorhinis was 27–28°C lower than that of M. fermentans. Due to PNA's high specificity, this larger melting temperature gap is easy to create. FMCA using PNA offers an alternative method for specific DNA detection. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:730–735, 2015     

A noninvasive method for distinguishing among canid species: amplification and enzyme restriction of DNA from dung

Endangered San Joaquin kit foxes Vulpes macrotis mutica can be sympatrically distributed with as many as four other canids: red fox, gray fox, coyote and domestic dog. Canid scats are often found during routine fieldwork, but cannot be reliably identified to species. To detect and study the endangered kit fox, we developed mitochondrial DNA markers that can be amplified from small amounts of DNA extracted from scats. We amplified a 412-bp fragment of the mitochondrial cytochrome- b gene from scat samples and digested it with three restriction enzymes. The resulting restriction profiles discriminated among all five canid species and correctly identified 10 'unknown' fox scats to species in blind tests. We have applied our technique to identify canids species for an environmental management study and a conservation study. We envision that our protocol, and similar ones developed for other endangered species will be greatly used for conservation management in the future.     

Discrimination of Mycobacterium avium- Mycobacterium intracellulare strains by genomic DNA fingerprinting with a 16S rRNA gene probe

Abstract Ribotyping was investigated as a means of distinguishing ten different serotyped reference strains and seven epidemiologically unrelated isolates of Mycobacterium avium - Mycobacterium intracellulare using a labelled 16S rDNA probe. Thirteen restriction enzymes were screened towards an accurate discrimination of strins. Two selected restriction enzymes ( Sac I and Cla I) enabled us to classify the 17 strains into ten ribotypes with an index of discrimination of 0.897. Typeability and reproductibility of the method reached 100%. The patterns obtained exhibited polymorphism of RE fragments within and outside the 16S rRNA gene and may be useful for epidemiological studies.     

A ribosomal DNA probe to distinguish populations of Rhopalosiphum maidis (Homoptera: Aphididae)

Total DNA from clones of Rhopalosiphum maidis was digested with restriction endonucleases and hybridised with a heterospecific ribosomal DNA probe. DNA banding patterns exhibited sufficient differences to distinguish five different clones, and provided greater resolving power than gel electrophoresis of allozymes. The mechanisms and applications of this marking technique are discussed.     

动物食性分析在生态学中的应用研究进展——基于DNA宏条形码技术

动物食性分析是动物营养生态学的重要研究手段,可用于解析动物与环境因素的关联性、捕食者与猎物之间的关系,以及动物物种多样性等科学问题。近年来,基于新一代测序技术的DNA宏条形码技术被广泛应用到生态学多个研究领域,极大地促进了生命科学交叉学科的发展。其中,DNA宏条形码技术在动物食性分析中具有高分辨、高效率、低样本量等优势,具有重要的应用前景。综述了基于DNA宏条形码技术的动物食性分析在生态学中的应用研究进展,并进一步总结了DNA宏条形码技术原理和食性分析方法,着重探讨了基于DNA宏条形码技术的动物食性分析在珍稀濒危动物保护、生物多样性监测、农业害虫防治等生态学研究领域中的应用,并对DNA宏条形码技术在动物食性分析中存在的问题及应用前景进行小结与展望。     

Wavelength shifting oligonucleotide probe for the detection of adenosine of a target DNA with enhanced fluorescence signal

The modulated photophysical property of strong electronically coupled naphthyl uridine linked via a single C–C bond was explored in DNA detection via wavelength shifting and enhanced fluorescence emission by a simple ‘Just-Mix & Read’ strategy of homogeneous DNA detection.     

DNA barcoding as a tool for early warning and monitoring alien duckweeds (Lemna sp.pl.): the case of Central Italy

Aquatic habitats are vulnerable to the invasion of alien species, so early warning protocols are necessary for eradication. The presence in Italy of two alien duckweeds in freshwaters has been documented: Lemna minuta, that showed high invasivity, and L. valdiviana, still confined to south Lazio. These two species may be mistaken for each other and for the domestic L. minor and L. gibba due to morphological variation. Here, we assess the applicability of DNA barcoding as a complement to morphological analysis for monitoring the spread of alien Lemna. We chose two chloroplast genome sequences for their ability to discriminate all Lemna species: the 5’ intron of the trnK gene and the matK gene. Among 48 samples of Lemna collected at 20 sites in Central Italy, 20 were identified as L. minor, 19 as L. minuta, five as L. trisulca and four as L. gibba. L. minuta was present at most sampling sites; in particular, at six locations of Lake Trasimeno, eight L. minuta samples were found. We demonstrate that DNA sequence analyses with cost-effective barcoding techniques can effectively support expert efforts in species determination for an early alert system of invasive Lemna species.     

A novel class of small repetitive DNA sequences in Enterococcus faecalis

The structural organization of Enterococcus faecalis repeats (EFAR) is described, palindromic DNA sequences identified in the genome of the Enterococcus faecalis V583 strain by in silico analyses. EFAR are a novel type of miniature insertion sequences, which vary in size from 42 to 650 bp. Length heterogeneity results from the variable assembly of 16 different sequence types. Most elements measure 170 bp, and can fold into peculiar L-shaped structures resulting from the folding of two independent stem-loop structures (SLSs). Homologous chromosomal regions lacking or containing EFAR sequences were identified by PCR among 20 E. faecalis clinical isolates of different genotypes. Sequencing of a representative set of 'empty' sites revealed that 24-37 bp-long sequences, unrelated to each other but all able to fold into SLSs, functioned as targets for the integration of EFAR. In the process, most of the SLS had been deleted, but part of the targeted stems had been retained at EFAR termini.     

Identification of fungal pathogens associated with grapevine trunk using fluorescent-labelled ribosomal DNA probe

Detection and identification of fungal pathogens associated with grapevine trunk diseases (GTDs) are often difficult and laborious. The aim of the study is to develop a simple and time-saving protocol for the identification of a broad range of fungal species causing GTDs by fragment length analysis of the internal transcribed spacer (ITS) regions 1 and 2 and the large subunit D2 hypervariable region of the ribosomal DNA (rDNA). A total of 32 fungal isolates from declining vines and six type strains, representing 19 different species was included in this study. The majority of the fungi had unique species-specific PCR products ranging from 212 to 370 bp (ITS1), 332 to 428 bp (ITS2) and 320 to 325 bp (LSU-D2), and the combination of three regions identifies all pathogenic fungi tested. The protocol proposed here provides a highly sensitive, reliable and rapid identification method for a broad range of significant grapevine fungal trunk pathogens.     

A novel environmental DNA approach to quantify the cryptic invasion of non‐native genotypes

The invasion of non‐native species that are closely related to native species can lead to competitive elimination of the native species and/or genomic extinction through hybridization. Such invasions often become serious before they are detected, posing unprecedented threats to biodiversity. A Japanese native strain of common carp (Cyprinus carpio) has become endangered owing to the invasion of non‐native strains introduced from the Eurasian continent. Here, we propose a rapid environmental DNA‐based approach to quantitatively monitor the invasion of non‐native genotypes. Using this system, we developed a method to quantify the relative proportion of native and non‐native DNA based on a single‐nucleotide polymorphism using cycling probe technology in real‐time PCR. The efficiency of this method was confirmed in aquarium experiments, where the quantified proportion of native and non‐native DNA in the water was well correlated to the biomass ratio of native and non‐native genotypes. This method provided quantitative estimates for the proportion of native and non‐native DNA in natural rivers and reservoirs, which allowed us to estimate the degree of invasion of non‐native genotypes without catching and analysing individual fish. Our approach would dramatically facilitate the process of quantitatively monitoring the invasion of non‐native conspecifics in aquatic ecosystems, thus revealing a promising method for risk assessment and management in biodiversity conservation.     

Synthesis of universal unmethylated control DNA by nested whole genome amplification with phi29 DNA polymerase

Optimization of highly sensitive methods to detect methylation of CpG islands in gene promoter regions requires adequate methylated and unmethylated control DNA. Whereas universal methylated control DNA is available, universal unmethylated control (UUC) DNA has not been made because demethylase is not available to remove methyl groups from all methylated cytosines. On the basis that DNA synthesized by DNA polymerase does not contain methylated cytosines, we developed a method to create UUC DNA by nested whole genome amplification (WGA) with phi29 DNA polymerase. Contamination of the template genomic DNA in UUC was only 3.1 x 10(-7), below the detection limit of sensitive methods used for methylation studies such as methylation-specific PCR. Assessment of microsatellite markers demonstrated that even nested phi29 WGA achieves highly accurate and homogeneous amplification with very low amounts of genomic DNA as an initial template. The UUC DNA created by nested phi29 WGA is practically very useful for methylation analysis.     

A hierarchical analysis of minisatellite DNA diversity in Gambel oak (Quercus gambelii Nutt.; Fagaceae)

Gambel oak is a short shrub to medium-sized tree of southwest North America for which the potential to grow in large clonal stands has been proposed. Here we use three different multilocus VNTR DNA probes (synthesized via PCR) to demonstrate that clones growing together can be identified, but individual clones exceeding 50 m in diameter do not commonly occur in the study population of Quercus gambelii . Further, using a hierarchical sampling scheme (three circular transects with diameters of 9, 1100, and 28 000 m), we estimate that for nonclonal individuals: (i) mean number of bands analysed per individual = 22.92; (ii) mean similarity (band sharing) between individuals = 0.322; (iii) mean probability that two randomly chosen individuals share all bands = 4.93 × 10^–11; and (iv) mean estimated heterozygosity = 0.796. F _ST calculated for the two nonclonal levels of the sampling hierarchy was 0.023, indicating that little genetic differentiation exists between them. These results support previous findings that, due to life-history traits of the genus and this species (out-crossing, wind-pollinated, animal-dispersed, long-lived woody perennials), gene flow is high and genetic subdivision of populations is low in oaks. At this study site, the clonal nature demonstrated for Gambel oak appears to have little detectable effect on these population genetic characteristics.     

A simple method for isolation of megabase DNA from cotton

A simple method for preparation of high-molecular-weight DNA from cotton was developed. This method includes two major steps, (i) isolating nuclei and (ii) embedding nuclei into agarose microbeads. DNA isolated by this procedure is larger than 5.7 Mb in size, and is suitable for physical mapping by PFGE and YAC/BAC cloning.     

Molecular differentiation of Bifidobacterium species with amplified ribosomal DNA restriction analysis and alignment of short regions of the ldh gene

The differentiation of Bifidobacterium species was performed with specific primers using the PCR technique, the amplified ribosomal DNA restriction analysis (ARDRA) technique based on reports on the sequence of the 16S rRNA gene and speciation based on a short region of the ldh gene. Four specific primer sets were developed for each of the Bifidobacterium species, B. animalis, B. infantis and B. longum. The use of the ARDRA method made it possible to discriminate between B. infantis, B. longum and B. animalis with the combination of BamHI, TaqI and Sau3AI restriction enzymes. The ldh gene sequences of 309-312 bp were determined for 19 Bifidobacterium strains. Alignment of these short regions of the ldh gene confirmed that it is possible to distinguish between B. longum and B. infantis but not between B. lactis and B. animalis.     

A Phylogenetic Analysis of Heterorhabditis (Nemata: Rhabditidae) Based on Internal Transcribed Spacer 1 DNA Sequence Data

Internal transcribed spacer 1 sequences were used to infer phylogenetic relationships among 8 of the 9 described species and one putative species of the entomopathogenic nematode genus Heterorhabditis. Sequences were aligned and optimized based on pairwise genetic distance and parsimony criteria and subjected to a variety of sequence alignment parameters. Phylogenetic trees were constructed with maximum parsimony, cladistic, distance, and maximum likelihood algorithms. Our results gave strong support for four pairs of sister species, while relationships between these pairs also were resolved but less well supported. The ITS1 region of the nuclear ribosomal repeat was a reliable source of homologous characters for resolving relationships between closely related taxa but provided more tenuous resolution among more divergent lineages. A high degree of sequence identity and lack of autapomorphic characters suggest that sister species pairs within three distinct lineages may be mutually conspecific. Application of these molecular data and current morphological knowledge to the delimitation of species is hindered by an incomplete understanding of their variability in natural populations.     

DYRK1A regulates the recruitment of 53BP1 to the sites of DNA damage in part through interaction with RNF169

Human Dual-specificity tyrosine (Y) Regulated Kinase 1A (DYRK1A) is encoded by a dosage dependent gene whereby either trisomy or haploinsufficiency result in developmental abnormalities. However, the function and regulation of this important protein kinase are not fully understood. Here, we report proteomic analysis of DYRK1A in human cells that revealed a novel role of DYRK1A in DNA double-strand breaks (DSBs) repair, mediated in part by its interaction with the ubiquitin-binding protein RNF169 that accumulates at the DSB sites and promotes homologous recombination repair (HRR) by displacing 53BP1, a key mediator of non-homologous end joining (NHEJ). We found that overexpression of active, but not the kinase inactive DYRK1A in U-2 OS cells inhibits accumulation of 53BP1 at the DSB sites in the RNF169-dependent manner. DYRK1A phosphorylates RNF169 at two sites that influence its ability to displace 53BP1 from the DSBs. Although DYRK1A is not required for the recruitment of RNF169 to the DSB sites and 53BP1 displacement, inhibition of DYRK1A or mutation of the DYRK1A phosphorylation sites in RNF169 decreases its ability to block accumulation of 53BP1 at the DSB sites. Interestingly, CRISPR-Cas9 knockout of DYRK1A in human and mouse cells also diminished the 53BP1 DSB recruitment in a manner that did not require RNF169, suggesting that dosage of DYRK1A can influence the DNA repair processes through both RNF169-dependent and independent mechanisms. Human U-2 OS cells devoid of DYRK1A display an increased HRR efficiency and resistance to DNA damage, therefore our findings implicate DYRK1A in the DNA repair processes.     

A parallel study of different array-CGH platforms in a set of Spanish patients with developmental delay and intellectual disability

Developmental delay and intellectual disability, which occur in 1–3% of the population, account for a large number of the cases regularly seen in genetic units. Chromosomal microarray analysis has been shown to be a valuable clinical diagnostic assay and it should be the first-tier clinical diagnostic test for individuals with these conditions. However and due to several difficulties such as the platform resolution, the cost, and the inexperience with genomic data bases, the implementation of this test in many cytogenetic laboratories has been delayed. In an attempt to provide more insights of the benefits derived by using the chromosomal microarray analysis, this study presents the experience of two clinical centers using three different microarray platforms. The results obtained using a custom microarray (KaryoArray®) and two different commercial medium- and high-resolution whole-genome oligonucleotide microarrays have been compared. An overall diagnostic yield of around 15% has been obtained. However, the custom microarray platform has been shown to be more convenient for a clinical setting, since it allows the detection of more pathogenic copy number variants and less common variants.