Tri-nucleotide threading for parallel amplification of minute amounts of genomic DNA

Efforts to correlate genetic variations with phenotypic differences are intensifying due to the availability of high-density maps of single nucleotide polymorphisms (SNPs) and the development of high throughput scoring methods. These recent advances have led to an increased interest for improved multiplex preparations of genetic material to facilitate such whole genome analyses. Here we propose a strategy for the parallel amplification of polymorphic loci based on a reduced set of nucleotides. The technique denoted Tri-nucleotide Threading (TnT), allows SNPs to be amplified via controlled linear amplification followed by complete removal of the target material and subsequent amplification with a pair of universal primers. A dedicated software tool was developed for this purpose and variable positions in genes associated with different forms of cancer were analyzed using sub-nanogram amounts of starting material. The amplified fragments were then successfully scored using a microarray-based PrASE technique. The results of this study, in which 75 SNPs were analyzed, show that the TnT technique circumvents potential problems associated with multiplex amplification of SNPs from minute amounts of material. The technique is specific, sensitive and can be readily adapted to equipment and genotyping techniques used in other research laboratories without requiring changes to the preferred typing method

Competitive enzymatic reaction to control allele-specific extensions

Here, we present a novel method for SNP genotyping based on protease-mediated allele-specific primer extension (PrASE), where the two allele-specific extension primers only differ in their 3′-positions. As reported previously [Ahmadian,A., Gharizadeh,B., O'Meara,D., Odeberg,J. and Lundeberg,J. (2001), Nucleic Acids Res., 29, e121], the kinetics of perfectly matched primer extension is faster than mismatched primer extension. In this study, we have utilized this difference in kinetics by adding protease, a protein-degrading enzyme, to discriminate between the extension reactions. The competition between the polymerase activity and the enzymatic degradation yields extension of the perfectly matched primer, while the slower extension of mismatched primer is eliminated. To allow multiplex and simultaneous detection of the investigated single nucleotide polymorphisms (SNPs), each extension primer was given a unique signature tag sequence on its 5′ end, complementary to a tag on a generic array. A multiplex nested PCR with 13 SNPs was performed in a total of 36 individuals and their alleles were scored. To demonstrate the improvements in scoring SNPs by PrASE, we also genotyped the individuals without inclusion of protease in the extension. We conclude that the developed assay is highly allele-specific, with excellent multiplex SNP capabilities

Gene-specific FACS sorting method for target selection in high-throughput amplicon sequencing

Background In addition to shotgun sequencing, next generation sequencing has been shown to be suitable for deep sequencing of many specific PCR-amplified target genes in parallel. However, unspecific product formation is a common problem in amplicon sequencing since these fragments are difficult to fully remove by gel purification, and their presence inevitably reduces the number of mappable sequence reads that can be obtained in each sequencing run. Results We have used a novel flow cytometric sorting approach to specifically enrich Roche/454 DNA Capture beads carrying target DNA sequences on their surface, and reject beads carrying unspecific sequences. This procedure gives a nearly three-fold increase in the fraction of informative sequences obtained. Presented results also show that there are no significant differences in the distribution or presence of different genotypes between a FACS-enriched sample and a standard-enriched control sample. Conclusions Target-specific FACS enrichment prior to Roche/454 sequencing provides a quick, inexpensive way of increasing the amount of high quality data obtained in a single sequencing run, without introducing any sequence bias.QC 2010090

Top down strategy for renewable energy investment: sizing methodologies and Integrated Renewable Energy System models

In this paper, we present a top down strategy for renewable energy investment. The proposed approach is a three-step framework. By applying the approach, renewable energy global market leaders and trends will be identified and analyzed that included: (1) economics and renewable energy policy, (2) specific renewable energy sectors that presents the most attractive investment opportunity, (3) and finally the most promising renewable energy investment vehicles for investors. Other stakeholders can also use the developed framework, such as consumers and policymakers, to make socio-economic decisions and assess renewable energy investments. This paper presents an extensive review on various issues related to Integrated Renewable Energy System (IRES) based power generation. Issues related to integration configurations, storage options, sizing methodologies and system control for energy flow management are discussed in detail

Narrow genetic basis for the Australian dingo confirmed through analysis of paternal ancestry

The dingo (Canis lupus dingo) is an iconic animal in the native culture of Australia, but archaeological and molecular records indicate a relatively recent history on the continent. Studies of mitochondrial DNA (mtDNA) imply that the current dingo population was founded by a small population of already tamed dogs from Southeast Asia. However, the maternal genetic data might give a unilateral picture, and the gene pool has yet to be screened for paternal ancestry. We sequenced 14,437 bp of the Y-chromosome (Y-chr) from two dingoes and one New Guinea Singing Dog (NGSD). This positioned dingo and NGSD within the domestic dog Y-chr phylogeny, and produced one haplotype not detected before. With this data, we characterized 47 male dingoes in 30 Y-chr single-nucleotide polymorphism sites using protease-mediated allele-specific extension technology. Only two haplotypes, H3 and H60, were found among the dingoes, at frequencies of 68.1 and 31.9 %, respectively, compared to 27 haplotypes previously established in the domestic dog. While H3 is common among Southeast Asian dogs, H60 was specifically found in dingoes and the NGSD, but was related to Southeast Asian dog Y-chr haplotypes. H3 and H60 were observed exclusively in the western and eastern parts of Australia, respectively, but had a common range in Southeast. Thus, the Y-chr diversity was very low, similar to previous observations for d-loop mtDNA. Overall genetic evidence suggests a very restricted introduction of the first dingoes into Australia, possibly from New Guinea. This study further confirms the dingo as an isolated feral dog. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s10709-012-9658-5) contains supplementary material, which is available to authorized users

A new hybrid algorithm for multi‐objective reactive power planning via facts devices and renewable wind resources

The power system planning problem considering system loss function, voltage profile function, the cost function of FACTS (flexible alternating current transmission system) devices, and stability function are investigated in this paper. With the growth of electronic technologies, FACTS devices have improved stability and more reliable planning in reactive power (RP) planning. In addition, in modern power systems, renewable resources have an inevitable effect on power system planning. Therefore, wind resources make a complicated problem of planning due to conflicting functions and non-linear constraints. This confliction is the stochastic nature of the cost, loss, and voltage functions that cannot be summarized in function. A multi-objective hybrid algorithm is proposed to solve this problem by considering the linear and non-linear constraints that combine particle swarm optimization (PSO) and the virus colony search (VCS). VCS is a new optimization method based on viruses’ search function to destroy host cells and cause the penetration of the best virus into a cell for reproduction. In the proposed model, the PSO is used to enhance local and global search. In addition, the non-dominated sort of the Pareto criterion is used to sort the data. The optimization results on different scenarios reveal that the combined method of the proposed hybrid algorithm can improve the parameters such as convergence time, index of voltage stability, and absolute magnitude of voltage deviation, and this method can reduce the total transmission line losses. In addition, the presence of wind resources has a positive effect on the mentioned issue

Flow cytometry for enrichment and titration in massively parallel DNA sequencing

Massively parallel DNA sequencing is revolutionizing genomics research throughout the life sciences. However, the reagent costs and labor requirements in current sequencing protocols are still substantial, although improvements are continuously being made. Here, we demonstrate an effective alternative to existing sample titration protocols for the Roche/454 system using Fluorescence Activated Cell Sorting (FACS) technology to determine the optimal DNA-to-bead ratio prior to large-scale sequencing. Our method, which eliminates the need for the costly pilot sequencing of samples during titration is capable of rapidly providing accurate DNA-to-bead ratios that are not biased by the quantification and sedimentation steps included in current protocols. Moreover, we demonstrate that FACS sorting can be readily used to highly enrich fractions of beads carrying template DNA, with near total elimination of empty beads and no downstream sacrifice of DNA sequencing quality. Automated enrichment by FACS is a simple approach to obtain pure samples for bead-based sequencing systems, and offers an efficient, low-cost alternative to current enrichment protocols

Screening of common CYP1B1 mutations in Iranian POAG patients using a microarray-based PrASE protocol

Purpose: The gene coding cytochrome P4501B1 (CYP1B1) has been shown to be a major cause of primary congenital glaucoma in the Iranian population. More recently it was shown to also be important in juvenile-onset open angle glaucoma (JOAG). We aimed to further investigate the role of CYP1B1 in a larger cohort of primary open angle glaucoma (POAG) patients which included late-onset patients. We also aimed to set up a microarray based protocol for mutation screening with an intent of using the protocol in a future population level screening program. Methods: Sixty three POAG patients, nine affected family members, and thirty three previously genotyped primary congenital glaucoma (PCG) patients were included in the study. Clinical examination included slit lamp biomicroscopy, IOP measurement, gonioscopic evaluation, fundus examination, and measurement of perimetry. G61E, R368H, R390H, and R469W were screened by a protocol that included multiplexed allele specific amplification in the presence of a protease (PrASE), use of sequence tagged primers, and hybridization to generic arrays on microarray slides. The entire coding sequences of CYP1B1 and myocilin (MYOC) genes were sequenced in all individuals assessed by the microarray assay to carry a mutation. Intragenic single nucleotide polymorphism (SNP) haplotpes were determined for mutated alleles. Results: Genotypes assessed by the array-based PrASE methodology were in 100 concordance with sequencing results. Seven mutation carrying POAG patients (11.1) were identified, and their distribution was quite skewed between the juvenile-onset individuals (5/21) as compared to late-onset cases (2/42). Four of the seven mutation carrying Iranian patients harbored two mutated alleles. CYP1B1 mutated alleles in Iranian PCG and POAG patients shared common haplotypes. MYOC mutations were not observed in any of the patients. Conclusions: The PrASE approach allowed reliable simultaneous genotyping of many individuals. It can be an appropriate tool for screening common mutations in large sample sizes. The results suggest that CYP1B1 is implicated in POAG among Iranians, notably in the juvenile-onset form. Contrary to POAG patients studied in other populations, many mutation harboring Iranian patients carry two mutated alleles. We propose an explanation for this observation. © 2008 Molecular Vision

Analysis of Short Tandem Repeats by Parallel DNA Threading

The majority of studies employing short tandem repeats (STRs) require investigation of several of these genetic markers. As such, we demonstrate the feasibility of the trinucleotide threading (TnT) approach for scalable analysis of STRs. The TnT method represents a parallel amplification alternative that addresses the obstacles associated with multiplex PCR. In this study, analysis of the STR fragments was performed with capillary gel electrophoresis; however, it should be possible to combine our approach with the massive 454 sequencing platform to considerably increase the number of targeted STRs