NTMG (N-terminal Truncated Mutants Generator for cDNA): an automatic multiplex PCR assays design for generating various N-terminal truncated cDNA mutants

The sequential deletion method is generally used to locate the functional domain of a protein. With this method, in order to find the various N-terminal truncated mutants, researchers have to investigate the ATG-like codons, to design various multiplex polymerase chain reaction (PCR) forward primers and to do several PCR experiments. This web server (N-terminal Truncated Mutants Generator for cDNA) will automatically generate groups of forward PCR primers and the corresponding reverse PCR primers that can be used in a single batch of a multiplex PCR experiment to extract the various N-terminal truncated mutants. This saves much time and money for those who use the sequential deletion method in their research. This server is available at http://oblab.cs.nchu.edu.tw:8080/WebSDL/

Genome-wide selection of unique and valid oligonucleotides

Functional genomics methods are used to investigate the huge amount of information contained in genomes. Numerous experimental methods rely on the use of oligo- or polynucleotides. Nucleotide strand hybridization forms the underlying principle for these methods. For all these techniques, the probes should be unique for analyzed genes. In addition to being unique for the studied genes, the probes should fulfill a large number of criteria to be usable and valid. The criteria include for example, avoidance of self-annealing, suitable melting temperature and nucleotide composition. We developed a method for searching unique and valid oligonucleotides or probes for genes so that there is not even a similar (approximate) occurrence in any other location of the whole genome. By using probe size 25, we analyzed 17 complete genomes representing a wide range of both prokaryotic and eukaryotic organisms. More than 92% of all the genes in the investigated genomes contained valid oligonucleotides. Extensive statistical tests were performed to characterize the properties of unique and valid oligonucleotides. Unique and valid oligonucleotides were relatively evenly distributed in genes except for the beginning and end, which were somewhat overrepresented. The flanking regions in eukaryotes were clearly underrepresented among suitable oligonucleotides. In addition to distributions within genes, the effects on codon and amino acid usage were also studied

Analisis basa nukleotida dan desain primer pada intron gen amelogenin harimau sumatra (panthera tigris sumatrae)

Pita hasil amplifikasi AMELX dan AMELY sampel harimau sumatra jantan sulit dibedakan karena perbedaan berat molekul DNAnya hanya 20 pasang basa (bp). Kesulitan ini berdampak pada terjadinya kesalahan saat mendeteksi sampel individu jantan dan betina. Sehingga perlu untuk dilakukan analisis basa nukleotida AMELX dan AMELY harimau sumatra serta desain primer khusus untuk identifikasi jenis kelamin harimau sumatra. Tujuan penelitian ini adalah untuk menganalisis urutan basa nukleotida pada intron gen amelogenin harimau sumatra, mendesain primer untuk identifikasi jenis kelamin harimau sumatra berdasarkan urutan basa nukleotida pada intron gen amelogenin, dan membandingkan primer hasil desain dengan primer yang pernah dilaporkan sebelumnya. Metode dalam penelitian ini adalah deskriptif dengan observasi secara molekuler sekuen AMELX dan AMELY harimau sumatra. Sampel hasil amplifikasi di sekuensing dan dilakukan desain primer berdasarkan sekuen sampel. Diketahui bahwa panjang sekuen AMELX dan AMELY harimau sumatra masing-masing adalah 215 bp dan 194 bp dengan delesi 21 bp pada sekuen AMELY. Primer didesain berdasarkan sekuen AMELY harimau sumatra dan berhasil mengamplifikasi AMELY pada sampel harimau sumatra. Diketahui bahwa primer hasil desain lebih spesifik dibanding primer yang dilaporkan sebelumnya

Multiplex PCR primer design for gene family using genetic algorithm

The multiplex PCR experiment is to amplify multiple regions of a DNA sequence at the same time by using different primer pairs. Designing feasible primer pairs for multiplex PCR is a tedious task since there are too many constraints to be satisfied. In this paper, a new method for multiplex PCR primer design strategy using genetic algorithm is proposed. The proposed algorithm is able to find a set of suitable primer pairs more efficient and uses a MAP model to speed up the examination of the specificity constraint that is important for gene family sequences. The dry-dock experiment shows that the proposed algorithm finds several sets of primer pairs of gene family sequences for multiplex PCR that not only obey the design properties, but also have specificity

Genetic assessment of plant material for studies of the genetic causes to female biased sex ratios in Salix viminalis L.

Biased sex ratios are highly common among dioecious plants. Salix viminalis is a dioecious tree or a woody shrub that has an overall female biased sex ratio. Most likely the bias is initiated in an early stage of development either by abortion of certain male zygotes or by genetic incompatibility leading to failure of fusion between the sperm and the egg. Salix viminalis has a female heterogametic or ZW sexual system, where the W represents a female-specific chromosome or region, meaning that all female offspring has one maternally inherited haplotype. In this work, I designed primers for markers located in the sex determination region on chromosome 15. By changing different parameters of PCR and by analysing the sequences of all the individuals, the expected genotypes of the used individuals were confirmed. Thus, in the further studies the markers can be applied in offspring and their genotypes can be determined, in order to estimate the sex ratio. Moreover, a germination test of different crosses of two populations of S. viminalis was done. One population had even sex ratio and other one female biased sex ratio. By comparing the germination rates between the crosses, the effect of inbreeding on germination rates in each cross was studied. Furthermore, based on the germination test the relatedness between homozygosity and white seeds phenomena was studied, since we assume that white seeds have homozygous genotype. In addition, the data which included germination rates was analysed in JMP program using ANOVA test. The results indicate that the reasons that some PCR programs in this study gave better results using the same primers than the others may be formation of primer dimers, secondary structures and that the sex determination region has many repetitions. The results show that there is significant difference between the germination rates between the crosses and between the catkins of each cross. Based on assumptions, in this work it is suggested that white seeds may not present homozygous individuals. Moreover, homozygosity may be related also to the black seeds or the homozygous seeds might not be even fully formed. Furthermore, it is possible that genetic incompatibility between homozygous allelic combinations have led to the differences among the germination rates of the crosses. In the further studies of the white seeds phenomena, sequencing of the genomes of the seeds is required in order to investigate the relatedness between homozygosity and white seeds

Characterising food web responses to climate change using a combination of traditional and molecular tools

Freshwater ecosystems are considered hot spots for biodiversity and provide a wide range of ecosystem services for human beings. A variety of natural and anthropogenic stressors are now threatening the stability and prosperity of these ecosystems. In particular, climate change and pollution are the main stressors impacting all freshwater ecosystems on Earth. In this mix of multiple stressors, climate change is already having profound impacts, and is predicted to result in large-scale population collapses, species range shifts, and local species extinctions, as well as altered ecosystem properties. Scientists have spent considerable efforts in recent years investigating how these perturbations might impact food web structures and dynamics to predict potential future scenarios, but much of this work has been hindered by the slow pace of data generation using traditional techniques. Thus, there is a need to adopt and develop new approaches that can answer questions and generate data at a much higher pace. Molecular tools can address this issue by generating millions of DNA sequences in a short period of time with the potential to build food webs in a very reliable way. Therefore, a detailed understanding of food webs and their interactions is critical to predict what effects climate change will have in the near future, and we need to find faster and cheaper ways of building the necessary evidence base. This will ultimately improve our ability to forecast how communities and ecosystems will respond to global change and anticipate which species (and systems) are more likely to deteriorate under these new conditions. In this thesis, I have used a combination of traditional and molecular tools to characterize the diet of a widely distributed generalist predator. DNA sequencing revealed a higher number of links compared to traditional microscopy, but protocols need to be refined to accurately quantify each link. In addition to this, I carried out two sets of laboratory experiments to quantify warming impacts on freshwater invertebrate interactions. Functional response experiments showed increased feeding rates with warming, while qPCR was not able to detect changes in DNA retention time in predator gut contents.Open Acces

Biological Nanowires: Integration of the silver(I) base pair into DNA with nanotechnological and synthetic biological applications

Modern computing and mobile device technologies are now based on semiconductor technology with nanoscale components, i.e., nanoelectronics, and are used in an increasing variety of consumer, scientific, and space-based applications. This rise to global prevalence has been accompanied by a similarly precipitous rise in fabrication cost, toxicity, and technicality; and the vast majority of modern nanotechnology cannot be repaired in whole or in part. In combination with looming scaling limits, it is clear that there is a critical need for fabrication technologies that rely upon clean, inexpensive, and portable means; and the ideal nanoelectronics manufacturing facility would harness micro- and nanoscale fabrication and self-assembly techniques. The field of molecular electronics has promised for the past two decades to fill fundamental gaps in modern, silicon-based, micro- and nanoelectronics; yet molecular electronic devices, in turn, have suffered from problems of size, dispersion and reproducibility. In parallel, advances in DNA nanotechnology over the past several decades have allowed for the design and assembly of nanoscale architectures with single-molecule precision, and indeed have been used as a basis for heteromaterial scaffolds, mechanically-active delivery mechanisms, and network assembly. The field has, however, suffered for lack of meaningful modularity in function: few designs to date interact with their surroundings in more than a mechanical manner. As a material, DNA offers the promise of nanometer resolution, self-assembly, linear shape, and connectivity into branched architectures; while its biological origin offers information storage, enzyme-compatibility and the promise of biologically-inspired fabrication through synthetic biological means. Recent advances in DNA chemistry have isolated and characterized an orthogonal DNA base pair using standard nucleobases: by bridging the gap between mismatched cytosine nucleotides, silver(I) ions can be selectively incorporated into the DNA helix with atomic resolution. The goal of this thesis is to explore how this approach to “metallize” DNA can be combined with structural DNA nanotechnology as a step toward creating electronically-functional DNA networks. This work begins with a survey of applications for such a transformative technology, including nanoelectronic component fabrication for low-resource and space-based applications. We then investigate the assembly of linear Ag+-functionalized DNA species using biochemical and structural analyses to gain an understanding of the kinetics, yield, morphology, and behavior of this orthogonal DNA base pair. After establishing a protocol for high yield assembly in the presence of varying Ag+ functionalization, we investigate these linear DNA species using electrical means. First a method of coupling orthogonal DNA to single-walled carbon nanotubes (SWCNTs) is explored for self-assembly into nanopatterned transistor devices. Then we carry out scanning tunneling microscope (STM) break junction experiments on short polycytosine, polycationic DNA duplexes and find increased molecular conductance of at least an order of magnitude relative to the most conductive DNA analog. With an understanding of linear species from both a biochemical and nanoelectronic perspective, we investigate the assembly of nonlinear Ag+-functionalized DNA species. Using rational design principles gathered from the analysis of linear species, a de novo mathematical framework for understanding generalized DNA networks is developed. This provides the basis for a computational model built in Matlab that is able to design DNA networks and nanostructures using arbitrary base parity. In this way, DNA nanostructures are able to be designed using the dC:Ag+:dC base pair, as well as any similar nucleobase or DNA-inspired system (dT:Hg2+:dT, rA:rU, G4, XNA, LNA, PNA, etc.). With this foundation, three general classes of DNA tiles are designed with embedded nanowire elements: single crossover Holliday junction (HJ) tiles, T-junction (TJ) units, and double crossover (DX) tile pairs and structures. A library of orthogonal chemistry DNA nanotechnology is described, and future applications to nanomaterials and circuit architectures are discussed