Telomeric motifs are present in a highly repetitive element in the Plasmodium berghei genome

Using as probes the subfragments of the telomeric sequence previously cloned by us from Plasmodium berghei DNA, we identified and cloned a 2.3 kb repeat, largely overlapping the original telomeric insert. Restriction mapping indicated that cloned inserts (2.3 kb in length) represented circularly permutated versions of a rather well conserved repeated element, at least in part organized in tandem. The 2.3 kb repeat family with a copy number of about 300 occupies about 4% of the whole genome. The copies are unevenly distributed among the chromosome-sized molecules revealed by pulsed field gradient electrophoresis. Complete sequence determination of the 2.3 kb element revealed that telomere-related motifs are present with a characteristic pattern in a set of tandem repeats, 27 bp long. The perfect conservation of these motifs as well as the pattern of chromosomal distribution suggest that we are dealing with a specialised structure subject to selective mechanisms of amplification and maintenance.     

Effect of Flow on Complex Biological Macromolecules in Microfluidic Devices

Understanding the transport, orientation, and deformation of biological macromolecules by flow is important in designing microfluidic devices. In this study, epi-fluorescence microscopy was used to characterize the behavior of macromolecules in flow in a microfluidic device, particularly how the flow affects the conformation of the molecules. The microfluidic flow path consists of a large, inlet reservoir connected to a long, rectangular channel followed by a large downstream reservoir. The flow contains both regions of high elongation (along the centerline as the fluid converges from the upstream reservoir into the channel) and shear (in the channel near the walls). Solutions of -DNA labeled with a fluorescent probe were first characterized rheologically to determine fluid relaxation times, then introduced into the microfluidic device. Images of the DNA conformation in the device were captured through an epi-fluorescent microscope. The conformation of DNA molecules under flow showed tremendous heterogeneity, as observed by Chu [7,12] and co-workers in pure shear and pure elongational flows. Histograms of the distribution of conformations were measured along the channel centerline as a function of axial position and revealed dramatic stretching of the molecules due to the converging flow followed by an eventual return to equilibrium coil size far downstream of the channel entry. The importance of shear was probed via a series of measurements near the channel centerline and near the channel wall. High shear rates near the channel wall also resulted in dramatic stretching of the molecules, and may result in chain scission of the macromolecules.     

Optical mapping of Plasmodium falciparum chromosome 2

Detailed restriction maps of microbial genomes are a valuable resource in genome sequencing studies but are toilsome to construct by contig construction of maps derived from cloned DNA. Analysis of genomic DNA enables large stretches of the genome to be mapped and circumvents library construction and associated cloning artifacts. We used pulsed-field gel electrophoresis purified Plasmodium falciparum chromosome 2 DNA as the starting material for optical mapping, a system for making ordered restriction maps from ensembles of individual DNA molecules. DNA molecules were bound to derivatized glass surfaces, cleaved with NheI or BamHI, and imaged by digital fluorescence microscopy. Large pieces of the chromosome containing ordered DNA restriction fragments were mapped. Maps were assembled from 50 molecules producing an average contig depth of 15 molecules and high-resolution restriction maps covering the entire chromosome. Chromosome 2 was found to be 976 kb by optical mapping with NheI, and 946 kb with BamHI, which compares closely to the published size of 947 kb from large-scale sequencing. The maps were used to further verify assemblies from the plasmid library used for sequencing. Maps generated in silico from the sequence data were compared to the optical mapping data, and good correspondence was found. Such high-resolution restriction maps may become an indispensable resource for large-scale genome sequencing projects.     

The human immunoglobulin VH7 gene family consists of a small,polymorphic group of six to eight gene segments dispersed throughout the VH locus

In this report we describe the analysis and mapping of members of the human immunoglobulin V_H7 gene family. V_H7 and V_H1 gene segments are closely related, with individual gene segments sharing between 78% and 82% sequence identity. Divergence from V_H1 gene sequence occurs as an abrupt event at the boundary between framework region (FR) 2 and complementarity-determining region (CDR) 2 and continues through a major portion of FR 3. We used polymerase chain reaction amplification to create a 162-base pair probe spanning the family-specific region of CDR 2 and FR 3 that proved suitable for standard Southern analysis of genomic DNA. The V_H7 gene family was found to be a small but discrete V_H gene family consisting of five to eight germ-line elements, of which at least three are polymorphic. Four different V_H7 gene segments were cloned from the germ line of a single individual, and assigned to specific restriction fragments by sequence-specific hybridization. Two of the four V_H7 elements were pseudogenes. The pattern of sequence variation in these and other known pseudogenes suggests that these nonfunctional elements may play a role in the evolution of novel V_H families. A combination of one and two-dimensional pulsed field gel electrophoresis was employed to map the chromosomal location of all of these V_H7 elements. Individual V_H7 gene segments were found to be dispersed over a region of at least 940 kb of DNA, and interspersed with members from other V_H gene families. The polymorphism of the V_H7 gene segments and their scattered location throughout the V_H locus makes them potentially useful markers for mapping and linkage studies.     

Xdrop: Targeted sequencing of long DNA molecules from low input samples using droplet sorting

Long‐read sequencing can resolve regions of the genome that are inaccessible to short reads, and therefore are ideal for genome‐gap closure, solving structural rearrangements and sequencing through repetitive elements. Here we introduce the Xdrop technology: a novel microfluidic‐based system that allows for targeted enrichment of long DNA molecules starting from only a few nanograms of DNA. Xdrop is based on the isolation of long DNA fragments in millions of droplets, where the droplets containing a target sequence of interest are fluorescently labeled and sorted using flow cytometry. The final product from the Xdrop procedure is an enriched population of long DNA molecules that can be investigated by sequencing. To demonstrate the capability of Xdrop, we performed enrichment of the human papilloma virus 18 integrated into the genome of human HeLa cells. Analysis of the sequencing reads resolved three HPV18‐chr8 integrations at base‐pair resolution, and the captured fragments extended up to 30 kb into the human genome at the integration sites. Further, we enriched the complete TP53 locus in a leukemia cell line and could successfully phase coexisting mutations using PacBio sequencing. In summary, our results show that Xdrop is an efficient enrichment technology for studying complex genomic regions.     

DNA motifs suppressing TLR9 responses

Immune cells respond to bacterial DNA containing unmethylated CpG motifs via Toll-like receptor 9 (TLR9). Given the apparent role of TLR9 in development of systemic lupus erythematosus (SLE), there is interest in the development of TLR9 inhibitors. TLR9-mediated responses are reported to be inhibited by a confusing variety of different DNA sequences and structures. To aid characterization, we have provisionally categorized TLR9-inhibitory oligodeoxynucleotides (ODN) into 4 classes, on the basis of sequence and probable mode of action. Class I are short G-rich ODN, which show sequence-specific inhibition of all TLR9 responses, and may be direct competitive inhibitors for DNA binding to TLR9. Class II are telomeric repeat motifs that inhibit STAT signaling, and thus are not specific to TLR9 responses. Because Class II ODN are generally made as 24-base phosphorothioate-modified ODN (PS-ODN), they also fall into Class IV, defined as long PS-ODN, which inhibit TLR9 responses in a sequence-nonspecific manner. Class III includes oligo (dG) that forms a 4-stranded structure and inhibits DNA uptake. The Class I G-rich motifs show the most promise as selective and potent TLR9 inhibitors for therapeutic applications.     

PCR amplification introduces errors into mononucleotide and dinucleotide repeat sequences.

The polymerase chain reaction (PCR) is used universally for accurate exponential amplification of DNA. We describe a high error rate at mononucleotide and dinucleotide repeat sequence motifs. Subcloning of PCR products allowed sequence analysis of individual DNA molecules from the product pool and revealed that: (1) monothymidine repeats longer than 11 bp are amplified with decreasing accuracy, (2) repeats generally contract during PCR because of the loss of repeat units, (3) Taq and proofreading polymerase Pfu generate similar errors at mononucleotide and dinucleotide repeats, and (4) unlike the parent PCR product pool, individual clones containing a single repeat length produce no "shadow bands". These data demonstrate that routine PCR amplification alters mononucleotide and dinucleotide repeat lengths. Such sequences are common components of genetic markers, disease genes, and intronic splicing motifs, and the amplification errors described here can be mistaken for polymorphisms or mutations.     

Single oligonucleotide nested PCR: a rapid method for the isolation of genes and their flanking regions from expressed sequence tags

We report on the development of a new PCR technique for the isolation of genomic fragments that flank known DNA sequences. This technique, single oligonucleotide nested (SON)-PCR, relies on only two amplification reactions with two or three nested sequence-specific primers. It allows the isolation of DNA regions located on either side of a known DNA sequence, with high specificity. DNA products of 2 kb in size can be generated that all contain one copy of the same primer at both ends. Sequence analysis of these products indicates that the binding of the primers to non-specific DNA sites mainly depends on their overall complementarity to the target sequence. Moreover, analysis shows that short extensions of the primers can occur during the first amplification reaction and that a 2-bp overlap between subsequent primers can target their annealing to their predecessors sequence. Ninety percent of the DNA products larger than 0.5 kb correspond to fragments of interest and we obtained successful results with various templates and primer sets. SON-PCR therefore seems a very efficient and widely applicable method for the rapid identification of large unknown DNA regions. Based on available expressed sequence tags, this technique was applied to isolate the palH and pacC genes of the phytopathogenic fungus Botrytis cinerea, with their 5 or 3 flanking regions.     

DNA序列数值映射方法的研究

DNA序列的数值映射是用数学方法、物理方法和数字信号处理方法分析生物分子序列首先要解决的问题。本文分析了现有8种DNA序列数值映射方法的特点和适应性。在此基础上,提出了一种基于DNA序列中碱基出现概率的数值映射方法。大多数蛋白编码序列具有3-碱基周期特性(周期-3性质)。借助于具有周期-3性质的DNA序列的频谱分析,比较了8种数值映射方法的优劣,并证实了新方法的有效性。计算机仿真结果表明,基于复域的映射方法无论从携带原有生物分子序列的信息量,还是数值映射后所得功率谱的效果均优于其它7种映射方法,而DNA序列新数值映射方法能够获得与复域法几乎相同的识别率。     

Modeling of a Microfluidic Channel in the Presence of an Electrostatic Induced Cross-Flow

Amongst the processes that have been implemented in microfluidic devices, electrophoretic transport of charged molecules, along microfluidic channels, is one of the most commonly found. However, less work has been done about continuous, pressure gradient driven flow systems where an electric field is applied orthogonally with respect to the microchannel walls. The perspective applications of this technique, include continuous flow separation and concentration of analyte molecules, and the kinetic control of surface reactions. In order to dimensioning and optimizing such a device, a mathematical model has been formulated and analyzed both with numeric and analytic methods. The given solutions let the designer of microfluidic devices able to estimate the concentration profiles along the microchannel length, as a function of the main system parameters. As a practical example of application which could be of great interest in biotechnology applications, the results relative to the simulation of the electrostatic induced cross flow of single strand DNA oligonucleotides of about 20 bases has been reported.     

Microfluidic gradient PCR (MG-PCR): a new method for microfluidic DNA amplification

This study develops a new microfluidic DNA amplification strategy for executing parallel DNA amplification in the microfluidic gradient polymerase chain reaction (MG-PCR) device. The developed temperature gradient microfluidic system is generated by using an innovative fin design. The device mainly consists of modular thermally conductive copper flake which is attached onto a finned aluminum heat sink with a small fan. In our microfluidic temperature gradient prototype, a non-linear temperature gradient is produced along the gradient direction. On the copper flake of length 45 mm, width 40 mm and thickness 4 mm, the temperature gradient easily spans the range from 97 to 52°C. By making full use of the hot (90–97°C) and cold (60–70°C) regions on the temperature gradient device, the parallel, two-temperature MG-PCR amplification is feasible. As a demonstration, the MG-PCR from three parallel reactions of 112-bp Escherichia coli DNA fragment is performed in a continuous-flow format, in which the flow of the PCR reagent in the closed loop is induced by the buoyancy-driven nature convection. Although the prototype is not optimized, the MG-PCR amplification can be completed in less than 45 min. However, the MG-PCR thermocycler presented herein can be further scaled-down, and thus the amplification times and reagent consumption can be further reduced. In addition, the currently developed temperature gradient technology can be applied onto other continuous-flow MG-PCR systems or used for other analytical purposes such as parallel and combination measurements, and fluorescent melting curve analysis.     

Magnetophoretic-based microfluidic device for DNA Concentration

Nucleic acids serve as biomarkers of disease and it is highly desirable to develop approaches to extract small number of such genomic extracts from human bodily fluids. Magnetic particles-based nucleic acid extraction is widely used for concentration of small amount of samples and is followed by DNA amplification in specific assays. However, approaches to integrate such magnetic particles based capture with micro and nanofluidic based assays are still lacking. In this report, we demonstrate a magnetophoretic-based approach for target-specific DNA extraction and concentration within a microfluidic device. This device features a large chamber for reducing flow velocity and an array of μ-magnets for enhancing magnetic flux density. With this strategy, the device is able to collect up to 95 % of the magnetic particles from the fluidic flow and to concentrate these magnetic particles in a collection region. Then an enzymatic reaction is used to detach the DNA from the magnetic particles within the microfluidic device, making the DNA available for subsequent analysis. Concentrations of over 1000-fold for 90 bp dsDNA molecules is demonstrated. This strategy can bridge the gap between detection of low concentration analytes from clinical samples and a range of micro and nanofluidic sensors and devices including nanopores, nano-cantilevers, and nanowires.     

Salmonella phage PSP3, another member of the P2-like phage group

Freshly isolated DNA of phage PSP3, whose morphology closely resembles that of phage P2, contained both circular and linear molecules about 31 kb in length. Linear PSP3 DNA molecules possess single-stranded cohesive termini (cos). Sequencing of the fragment anticipated to contain cos revealed a 19-base sequence identical to cos of phage 186. Of the 107 bp to the right of cos, 94 were identical in 186 DNA (88% similarity), and of the 370 bp to the left, 229 were identical (62% similarity). Cos flanking sequences in both P2 and P4 were also highly conserved in PSP3. A number of restriction sites were at similar locations on the two phage DNAs. The parasitic phage P4 propagated on PSP3 lysogens. PSP3 integrates into the Escherichia coli chromosome at 27 min.     

Transcriptome analysis device based on liquid phase detection by fluorescently labeled nucleic acid probes

Personalized medicine based on genetic information has been proposed as an attractive medical treatment. It is supported by the rapid development of the gene diagnosis utilizing on-chip analysis technology. This study reports characterizations of mRNA detection device by its hybridization with 2'-O-methyl oligoribonucleotide probe in a liquid phase, which eliminates time-consuming processes such as removing non-hybridized probes in conventional solid phase detection. In order to achieve a high sensitivity in the fluorescent detection of the target mRNA, we have optimized detection channel depth and width for a microfluidic device, and investigated polydimethyl siloxiane components and observation setup to minimize background noise. Adopting optimized channel dimensions and setup for the probe, the fluorescent intensity increased 3.3-fold at the lowest detectable concentration of 7.8 nM.