scholarly journals Sequencing of Genomic DNA by Combined Amplification and Cycle Sequencing Reaction

2005 ◽  
Vol 51 (1) ◽  
pp. 35-39 ◽  
Author(s):  
Kathleen M Murphy ◽  
Karin D Berg ◽  
James R Eshleman
Keyword(s):  
Genomic Dna ◽  
Direct Sequencing ◽  
Pcr Amplification ◽  
Target Sequence ◽  
Factor V ◽  
Cycle Sequencing ◽  
Bacterial Speciation ◽  
Set Up ◽  
The Cost ◽  
Single Reaction

Abstract Background: Despite considerable advances, DNA sequencing has remained somewhat time-consuming and expensive, requiring three separate steps to generate sequencing products from a template: amplification of the target sequence; purification of the amplified product; and a sequencing reaction. Our aim was to develop a method to routinely combine PCR amplification and cycle sequencing into one single reaction, enabling direct sequencing of genomic DNA. Methods: Combined amplification and sequencing reactions were set up with Big Dye™ sequencing reagents (Applied Biosystems) supplemented with variable amounts of forward and reverse primers, deoxynucleotide triphosphates (dNTPs), and input DNA. Reactions were thermal-cycled for 35 or 45 cycles. Products were analyzed by capillary electrophoresis to detect sequencing products. Results: Reactions using two oligonucleotide primers at a ratio of 5:1 (500 nM primer 1 and 100 nM primer 2), 125 μM supplemental dNTPs, and 35–45 thermal cycles optimally supported combined amplification and cycle sequencing reactions. Our results suggest that these reactions are dominated by PCR during early cycles and convert to cycle sequencing in later cycles. We used this technique for a variety of sequencing applications, including the identification of germline mutations/polymorphisms in the Factor V and BRCA2 genes, sequencing of tumor DNA to identify somatic mutations in the DPC4/SMADH4 and FLT3 genes, and sequencing of 16S ribosomal DNA for bacterial speciation. Conclusions: PCR amplification and cycle sequencing can be combined into a single reaction using the conditions described. This technique allows direct sequencing of genomic DNA, decreasing the cost and labor involved in gene sequencing.

Simple and Rapid Detection of Factor V Leiden by Allele-specific PCR Amplification

1996 ◽  
Vol 75 (05) ◽  
pp. 757-759 ◽  
Author(s):  
Rainer Blasczyk ◽  
Markus Ritter ◽  
Christian Thiede ◽  
Jenny Wehling ◽  
Günter Hintz ◽  
...  
Keyword(s):  
Direct Sequencing ◽  
Activated Protein C ◽  
Factor V Leiden ◽  
Pcr Amplification ◽  
Factor V ◽  
Specific Pcr ◽  
Pcr Rflp ◽  
Allele Specific ◽  
Specific Amplification ◽  
Allele Specific Pcr

SummaryResistance to activated protein C is the most common hereditary cause for thrombosis and significantly linked to factor V Leiden. In this study, primers were designed to identify the factor V mutation by allele-specific PCR amplification. 126 patients with thromboembolic events were analysed using this technique, PCR-RFLP and direct sequencing. The concordance between these techniques was 100%. In 27 patients a heterozygous factor VGln506 mutation was detected, whereas one patient with recurrent thromboembolism was homozygous for the point mutation. Due to its time- and cost-saving features allele-specific amplification should be considered for screening of factor VGln506.

scholarly journals Field and Laboratory Methods for DNA Studies on Deep-sea Isopod Crustaceans

2014 ◽  
Vol 35 (2) ◽  
pp. 203-224 ◽  
Author(s):  
Torben Riehl ◽  
Nils Brenke ◽  
Saskia Brix ◽  
Amy Driskell ◽  
Stefanie Kaiser ◽  
...  
Keyword(s):  
Deep Sea ◽  
Pcr Amplification ◽  
The Other ◽  
Universal Primers ◽  
Success Rates ◽  
Specific Primers ◽  
Scientific Publications ◽  
Cycle Sequencing ◽  
Laboratory Methods ◽  
Set Up

AbstractField and laboratory protocols that originally led to the success of published studies have previously been only briefly laid out in the methods sections of scientific publications. For the sake of repeatability, we regard the details of the methodology that allowed broad-range DNA studies on deep-sea isopods too valuable to be neglected. Here, a comprehensive summary of protocols for the retrieval of the samples, fixation on board research vessels, PCR amplification and cycle sequencing of altogether six loci (three mitochondrial and three nuclear) is provided. These were adapted from previous protocols and developed especially for asellote Isopoda from deep-sea samples but have been successfully used in some other peracarids as well. In total, about 2300 specimens of isopods, 100 amphipods and 300 tanaids were sequenced mainly for COI and 16S and partly for the other markers. Although we did not set up an experimental design, we were able to analyze amplification and sequencing success of different methods on 16S and compare success rates for COI and 16S. The primer pair 16S SF/SR was generally reliable and led to better results than universal primers in all studied Janiroidea, except Munnopsidae and Dendrotionidae. The widely applied universal primers for the barcoding region of COI are problematic to use in deep-sea isopods with a success rate of 45–79% varying with family. To improve this, we recommend the development of taxon-specific primers.

scholarly journals Detection of the Factor V Leiden Mutation by a Modified Photo-Cross-Linking Oligonucleotide Hybridization Assay

2004 ◽  
Vol 50 (2) ◽  
pp. 296-305 ◽  
Author(s):  
Cynthia French ◽  
Conan Li ◽  
Charles Strom ◽  
Weimin Sun ◽  
Reuel Van Atta ◽  
...  
Keyword(s):  
Genomic Dna ◽  
Factor V Leiden ◽  
Fold Increase ◽  
Clinical Samples ◽  
Cross Linking ◽  
Target Sequence ◽  
Factor V ◽  
Capture Probe ◽  
Factor V Leiden Mutation ◽  
Oligonucleotide Hybridization

Abstract Background: Our previously developed assay for detection of the factor V Leiden mutation (G1691A) based on a nucleic acid photo-cross-linking technology used two allele-specific capture probes and six fluorescein-modified signal-generating reporter probes. We wished to improve the sensitivity and performance of the method. Methods: We developed new reporter probes with ∼10-fold more fluorescein molecules than the original probes. The single, cross-linker-modified capture probe was replaced by a three-probe system, separating the probe–target cross-linking function and the allelic differentiation function. The capture probe cross-linked to either or both of two flanking probes through stem structures at the capture-probe/flanking-probe junctions. The flanking probes cross-linked to target DNA through two cross-linking sites each. Genomic DNA was extracted from 0.2 mL of whole blood and restriction-enzyme digested to create a defined 677 bp target sequence. Preliminary genotype ranges were determined for the assay by testing of pretyped samples. We then tested 1054 clinical samples, using an automated sample processor. Results: The new assay had a 10-fold increase in signal-to-background ratio. Genotype results for 1039 of 1054 clinical samples (98.6%) agreed with those of a PCR-based method. Of the 15 remaining samples, 10 produced an indeterminate result outside the defined genotype ranges, 2 yielded insufficient signal to be genotyped, and 3 gave a discordant result. All 15 samples were genotyped correctly after reextraction of genomic DNA and retesting. Conclusion: The modified photo-cross-linking assay for factor V Leiden detection is a sensitive non-PCR-based assay with potential for use in high-throughput clinical laboratories.

scholarly journals Lack of TEK Gene Mutation in Patients with Cutaneomucosal Venous Malformations from the North-Western Region of Algeria

2013 ◽  
Vol 2013 ◽  
pp. 1-7 ◽  
Author(s):  
Nabila Brahami ◽  
Mourad Aribi ◽  
Badr-Eddine Sari ◽  
Philippe Khau Van Kien ◽  
Isabelle Touitou ◽  
...  
Keyword(s):  
Genomic Dna ◽  
Direct Sequencing ◽  
Venous Malformation ◽  
Pcr Amplification ◽  
Venous Malformations ◽  
Dominant Form ◽  
The North ◽  
Vascular Morphogenesis ◽  
Western Algeria ◽  
North Western

Background. Venous malformations (VM) result from an error in vascular morphogenesis. The first gene suspected in their development is the TEK gene (tyrosine kinase, endothelial). Mutations of this gene have been identified in several Belgian families with a dominant form of the disease. Therefore, we investigated whether mutations in this TEK gene could explain the MV development in patients of families from Tlemcen region (north-western Algeria). Methods. Genomic DNA was extracted from leucocytes of ten patients. The search for mutations in all the 23 exons and in the 5′ and 3′ intronic sequences flanking the TEK gene was performed using PCR amplification and direct sequencing of amplified genomic DNA. Additionally, a search for somatic mutations of the gene TEK was performed on a biopsy of the venous malformation from one of the ten eligible patients. Results. The sequencing of the 23 exons of the TEK gene revealed neither germinal mutation in our ten patients nor somatic mutation in the tissue of the biopsy. Conclusion. The absence of mutation in the TEK gene in the population studied suggests that the TEK gene is not necessarily involved in the onset of VM; its association with these malformations may differ from one population to another.

Incidence and Clinical Significance of Nucleophosmin Mutations in Childhood AML: A Childrens Oncology Group Study.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 221-221 ◽  
Author(s):  
Patrick Brown ◽  
Emily McIntyre ◽  
Rachel Rau ◽  
Todd A. Alonzo ◽  
Robert Gerbing ◽  
...  
Keyword(s):  
Clinical Significance ◽  
Genomic Dna ◽  
Direct Sequencing ◽  
Prognostic Significance ◽  
Pcr Amplification ◽  
Normal Karyotype ◽  
Phase Iii ◽  
Remission Induction ◽  
Therapeutic Trial ◽  
Childhood Aml

Abstract Nucleophosmin mutations (NPM-mu) result in aberrant cytoplasmic localization of the NPM protein and occur in 25–35% of adult AML. NPM-mu are most commonly found in cases with normal karyotype, and are frequently associated with FLT3/ITD mutations. NPM-mu have been associated with high remission induction rates and improved survival, especially in patients with normal karyotype that lack FLT3/ITD mutations. The incidence and clinical significance of NPM-mu in childhood AML are less well-characterized. The AIEOP in Italy reported NPM-mu in 7 of 107 (6.5%) children treated on its AML02 protocol, and a Taiwanese group reported NPM-mu in 1 of 47 (2.1%) of children. The prognostic significance of NPM-mu in childhood AML is not known. The purpose of this study was to determine the incidence and clinical significance of NPM-mu in two large cohorts of children with newly-diagnosed AML treated on U.S. cooperative group phase III clinical trials (CCG-2961 and POG-9421). Criteria for selection of study patients included enrollment on the therapeutic trial and availability of banked genomic DNA (for CCG-2961) or RNA (for POG-9421). 919 patients met these criteria (566 from CCG-2961, 353 from POG-9421). For the genomic DNA samples, exon 12 of the nucleophosmin gene was directly amplified by PCR. The RNA samples were reverse transcribed to cDNA prior to PCR amplification. Mutations were detected using SSCP gel electrophoresis and confirmed by direct sequencing. The incidence of NPM-mu was 8.8% (9.5% for CCG, 7.7% for POG). As in prior reports, all of the mutations consisted of 4-bp insertions that resulted in changes in the 2 trytophan residues at AA positions 288 and 290 (important for nuclear localization). Only 48% of the mutations were of the “A” type (compared to 70–80% in adult AML), and 36% were novel mutations. FLT3/ITD mutations were more common in NPM-mu than NPM-wild type (wt) patients (17% vs. 9%, p=0.0381). NPM-mu patients were older than NPM-wt (median age 12 vs. 9 years, p=0.018). NPM-mu was particularly uncommon in children less than 3 years (1 mutation in 178 patients). Females accounted for 62% of the NPM-mut patients vs. 46% of the NPM-wt patients (p=0.0154). 73% of NPM-mut patients had normal cytogenetics, vs. 25% of NPM-wt patients (p<0.0001). There were no significant differences between NPM-mut and NPM-wt patients in median WBC, platelet or hemoglobin counts, FAB classification, hepatosplenomegaly or CNS disease. As shown in the table, there were no significant differences in EFS or OS for either cohort, although there was a trend towards improved survival for NPM-mu patients on POG 9421, particularly within the normal karyotype subset. In conclusion, NPM mutations are less common in children than adults and appear to have less prognostic relevance, although prospective studies will be needed to determine whether NPM-mu may contribute to risk stratification in children with normal karyotype. CCG-2961 N EFS OS NPM-mu 45 36% 49% NPM-wt 407 41% 53% logrank p=0.762 p=0.909 POG-9421 N EFS OS NPM-mu 23 48% 52% NPM-wt 270 33% 49% logrank p=0.284 p=0.660 POG-9421 (nl karyotype) N EFS OS NPM-mu 16 56% 56% NPM-wt 69 28% 39% logrank p=0.065 p=0.150

scholarly journals Single and Double Infections with Wolbachia in the Parasitic Wasp Nasonia vitripennis Effects on Compatibility

Genetics ◽  
1996 ◽  
Vol 143 (2) ◽  
pp. 961-972 ◽  
Author(s):  
Marie-Jeanne Perrot-Minnot ◽  
Li Rong Guo ◽  
John H Werren
Keyword(s):  
Genomic Dna ◽  
Rapid Development ◽  
Parasitic Wasp ◽  
Pcr Amplification ◽  
Bacterial Strains ◽  
Nasonia Vitripennis ◽  
Larval Diapause ◽  
Wide Range ◽  
Reproductive Incompatibility ◽  
Infected Line

Abstract Wolbachia are cytoplasmically inherited bacteria responsible for reproductive incompatibility in a wide range of insects. There has been little exploration, however, of within species Wolbachia polymorphisms and their effects on compatibility. Here we show that some strains of the parasitic wasp Nasonia vitripennis are infected with two distinct bacterial strains (A and B) whereas others are singly infected (A or B). Double and single infections are confirmed by both PCR amplification and Southern analysis of genomic DNA. Furthermore, it is shown that prolonged larval diapause (the overwintering stage of the wasp) of a double-infected strain can lead to stochastic loss of one or both bacterial strains. After diapause of a double-infected line, sublines were produced with AB, A only, B only or no Wolbachia. A and B sublines are bidirectionally incompatible, whereas males from AB lines are unidirectionally incompatible with females of A and B sublines. Results therefore show rapid development of bidirectional incompatibility within a species due to segregation of associated symbiotic bacteria.

The Mitigation of the French Nuclear Option: New Industrial Realism and Technical Democracy

2002 ◽  
Vol 13 (2) ◽  
pp. 263-279 ◽  
Author(s):  
Dominique Finon
Keyword(s):  
Waste Management ◽  
Electricity Markets ◽  
Social Preferences ◽  
Public Inquiry ◽  
The Future ◽  
Parliamentary Committee ◽  
Set Up ◽  
The Stability ◽  
The Cost ◽  
Phase Out

Nuclear phase-out policies and the European obligation to liberalise electricity markets could put the French nuclear option dramatically at risk by influencing social preferences or by constraining power producers' investment choices in the future. So far, the particular institutional set-up which has allowed the efficient build-up and operation of several series of standardised reactors preserves the stability of the main elements of the option. However, important adaptations to the evolving industrial and political environment occur and contribute to changing the option. Some institutional changes (such as local public inquiry, creation of a Parliamentary committee, independence of safety authorities) and divergence between industrial interests already allow debates on internal options such as reprocessing, type of waste management deposits, ordering of an advanced PWR. These changes improve the cost transparency, even if internalisation of nuclear externalities (cost of insurance, provisions for waste management) is still incomplete. However, when effective, this internalisation would not affect definitively the competitive position of the nuclear production because of the parallel internalisation of CO2 externalities from fossil fuel power generation in the official rationale. Consequently the real issue for the future of the nuclear option in France remains the preservation of social acceptability in the perception of nuclear risks.

Design for Variety: A Methodology for Understanding the Costs of Product Proliferation

1996 ◽  
Author(s):  
Mark V. Martin ◽  
Kosuke Ishii
Keyword(s):  
Indirect Costs ◽  
Product Line ◽  
Decision Makers ◽  
Profit Margins ◽  
Design For Variety ◽  
Set Up ◽  
Market Coverage ◽  
Product Proliferation ◽  
The Cost

Abstract This paper further develops the previously introduced concept of Design for Variety (DFV). Our study seeks a tool that enables product managers to estimate the cost of introducing variety into their product line. This will help them to maximize market coverage while maintaining required profit margins. Variety incurs many indirect costs that are not always well understood or are difficult to capture. These costs are often not considered by people making the decision about introducing variety. Our DFV model attempts to capture these indirect costs through the measurement of three indices: commonality, differentiation point, and set-up cost. These indices will allow the decision makers to estimate some of the generally unmeasurable costs of providing variety. We conclude this paper by discussing our validation plans for testing the model in industry.

Balqis restaurant: how to move on?

2017 ◽  
Vol 7 (3) ◽  
pp. 1-19
Author(s):  
Farzana Quoquab ◽  
Shazwani Binti Ahmad ◽  
Wan Nurul Syazwani Binti Wan Danial ◽  
Jihad Mohammad
Keyword(s):  
Subject Area ◽  
Marketing Mix ◽  
Business Success ◽  
Strategic Decisions ◽  
Gradual Improvement ◽  
Content Type ◽  
To Come ◽  
Set Up ◽  
The Right ◽  
The Cost

Subject area This case can be used in marketing management as well as consumer behaviour courses. Study level/applicability This case is suitable to use in advanced undergraduate levels, MBA and MSc in marketing courses that cover topics related to market segmentation and marketing mix strategies. Case overview This case highlights the dilemma of an entrepreneur and a manager of a restaurant who were to take a decision about the sustainability of their restaurant business. Balqis Restaurant was owned by Danny who was a retiree from Telekom Malaysia. He wanted to open a restaurant business after he came back from his long holiday trip. He conducted market research to find a suitable place to open his Arabic restaurant. He assigned Waleed Masood Abdullah as the manager of Balqis Restaurant. Finally, in June 2010, he opened his long awaited restaurant at Gombak, Kuala Lumpur. The restaurant was known as Qasar before the name was changed to Balqis in 2015 because of copyright issues related to Saba’ restaurant at Cyberjaya. The restaurant was well managed under Danny’s supervision for 4 years and successfully won customers’ hearts and loyalty before he decided to give full responsibility to Waleed in March 2014. Danny trusted Waleed because he taught and trained him. However, under Waleed’s management, Balqis started to lose its customers. Waleed also started to branch out the restaurant to different places in different states; one in Ipoh, and the other in Perak. He invested much money on renovation for all three branches, but one of the restaurants closed down in September 2014. This is because of the fact that they could no longer bear the cost of operations for the restaurant. However, he failed to learn from the mistake; they set up another restaurant, which was in Kuantan, in the same month. The sales were not that encouraging but it did show gradual improvement; yet, they once again sold it to another Arab businessman. Waleed realized his failure in managing the restaurant business in August 2015. He again opted to open another new branch which was questioned by Danny. He was in a rush to open it by the end of December 2015 to ensure that the additional profits from the current restaurants could cover the variables costs if the new restaurants were launched. Based on that, the owner had to make a decision about whether a new branch should be opened or whether they should just retain their restaurant in Gombak. Expected learning outcomes The learning objectives of using this case are as follows. 1. Knowledge enhancement: to help students in understanding the problems faced by a restaurant in expanding its market; to make students aware that a properly blended marketing mix is the key to business success and to broaden students’ views and understanding in targeting the proper market segment in formulating an effective marketing strategy. 2. Skills building: to be able to identify the best marketing strategic decisions to manage the restaurant business for its survival and to develop students’ ability to analyse the existing situation to come up with a viable and effective solution. 3. Attitudinal: to help the students to have intellectual openness in accepting different ways of finding solutions for a particular problem and to assist students in making the right move at the right time. Supplementary materials Teaching Notes are available for educators only. Please contact your library to gain login details or email [email protected] to request teaching notes. Subject code CSS 8: Marketing.

GAIN Premix Facility: An Innovative Approach for Improving Access to Quality Vitamin and Mineral Premix in Fortification Initiatives

2012 ◽  
Vol 33 (4_suppl3) ◽  
pp. S381-S389 ◽  
Author(s):  
Philippe Guinot ◽  
Vincent Jallier ◽  
Alessandro Blasi ◽  
Christophe Guyondet ◽  
Marc Van Ameringen
Keyword(s):  
Large Scale ◽  
Edible Oils ◽  
Quality Standards ◽  
Private Industry ◽  
Target Groups ◽  
High Quality ◽  
Adequate Quality ◽  
Standards And Guidelines ◽  
Set Up ◽  
The Cost

Background Vitamin and mineral premix is one of the most significant recurring input costs for large-scale food fortification programs. A number of barriers exist to procuring adequate quality premix, including accessing suppliers, volatile prices for premix, lack of quality assurance and monitoring of delivered products, and lack of funds to purchase premix. Objective To develop and test a model to procure premix through a transparent and efficient process in which an adequate level of quality is guaranteed and a financial mechanism is in place to support countries or specific target groups when there are insufficient resources to cover the cost of premix. Methods Efforts focused on premixes used to fortify flour, such as wheat or maize (iron, zinc, B vitamins, and vitamin A), edible oils (vitamins A and D), and other food vehicles, such as fortified complementary foods, complementary food supplements, and condiments. A premix procurement model was set up with three distinct components: a certification process that establishes industry-wide standards and guidelines for premix, a procurement facility that makes premix more accessible to countries and private industry engaged in fortification, and a credit facility mechanism that helps projects finance premix purchases. Results After three years of operation, 15 premix suppliers and 29 micronutrient manufacturers have been certified, and more than US$23 million worth of premix that met quality standards has been supplied in 34 countries in Africa, Central and Southern Asia, and Eastern Europe, reaching an estimated 242 million consumers. Conclusions The Premix Facility demonstrated its effectiveness in ensuring access to high-quality premixes, therefore enabling the success of various fortification programs.

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