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Results for “Primer design”

Search indexed NASA NTRS and DOE OSTI research on propulsion, heat transfer, battery materials and energy systems. Follow report and document links to the original sources.

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At least 19 records
All sourcesNASA NTRSDOE OSTI
DOE OSTI2014

PrimerDesign-M: A multiple-alignment based multiple-primer design tool for walking across variable genomes

Analyses of entire viral genomes or mtDNA requires comprehensive design of many primers across their genomes. In addition, simultaneous optimization of several DNA primer design criteria may improve overall experimental efficiency and downstream bioinformatic processing. To achieve these goals, we developed PrimerDesign-M. It includes several options for multiple-primer design, allowing researchers to efficiently design walking primers that cover long DNA targets, such as entire HIV-1 genomes, and that optimizes primers simultaneously informed by genetic diversity in multiple alignments and experimental design constraints given by the user. PrimerDesign-M can also design primers that include DNA barcodes and minimize primer dimerization. PrimerDesign-M finds optimal primers for highly variable DNA targets and facilitates design flexibility by suggesting alternative designs to adapt to experimental conditions.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2013

A multiple-alignment based primer design algorithm for genetically highly variable DNA targets

Background: Primer design for highly variable DNA sequences is difficult, and experimental success requires attention to many interacting constraints. The advent of next-generation sequencing methods allows the investigation of rare variants otherwise hidden deep in large populations, but requires attention to population diversity and primer localization in relatively conserved regions, in addition to recognized constraints typically considered in primer design. Results: Design constraints include degenerate sites to maximize population coverage, matching of melting temperatures, optimizing de novo sequence length, finding optimal bio-barcodes to allow efficient downstream analyses, and minimizing risk of dimerization. To facilitate primer design addressing these and other constraints, we created a novel computer program (PrimerDesign) that automates this complex procedure. We show its powers and limitations and give examples of successful designs for the analysis of HIV-1 populations. Conclusions: PrimerDesign is useful for researchers who want to design DNA primers and probes for analyzing highly variable DNA populations. It can be used to design primers for PCR, RT-PCR, Sanger sequencing, next generation sequencing, and other experimental protocols targeting highly variable DNA samples.

59 BASIC BIOLOGICAL SCIENCES↗
NASA NTRS1985

Design Primer for Reed-Solomon Encoders

Design and operation of Reed-Solomon (RS) encoders discussed in document prepared as instruction manual for computer designers and others in dataprocessing field. Conventional and Berlekamp architectures compared. Engineers who equip computer memory chips with burst-error and dropout detection and correction find report especially useful.

Perlman, M.↗
DOE OSTI2014

Multiplex Degenerate Primer Design for Targeted Whole Genome Amplification of Many Viral Genomes

Background . Targeted enrichment improves coverage of highly mutable viruses at low concentration in complex samples. Degenerate primers that anneal to conserved regions can facilitate amplification of divergent, low concentration variants, even when the strain present is unknown. Results . A tool for designing multiplex sets of degenerate sequencing primers to tile overlapping amplicons across multiple whole genomes is described. The new script, run_tiled_primers, is part of the PriMux software. Primers were designed for each segment of South American hemorrhagic fever viruses, tick-borne encephalitis, Henipaviruses, Arenaviruses, Filoviruses, Crimean-Congo hemorrhagic fever virus, Rift Valley fever virus, and Japanese encephalitis virus. Each group is highly diverse with as little as 5% genome consensus. Primer sets were computationally checked for nontarget cross reactions against the NCBI nucleotide sequence database. Primers for murine hepatitis virus were demonstrated in the lab to specifically amplify selected genes from a laboratory cultured strain that had undergone extensive passage in vitro and in vivo. Conclusions . This software should help researchers design multiplex sets of primers for targeted whole genome enrichment prior to sequencing to obtain better coverage of low titer, divergent viruses. Applications include viral discovery from a complex background and improved sensitivity and coverage of rapidly evolving strains or variants in a gene family.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Importance of appropriate genome information for the design of mating type primers in black and yellow morel populations

Abstract Morels are highly prized edible fungi where sexual reproduction is essential for fruiting-body production. As a result, a comprehensive understanding of their sexual reproduction is of great interest. Central to this is the identification of the reproductive strategies used by morels. Sexual reproduction in fungi is controlled by mating-type ( MAT ) genes and morels are thought to be mainly heterothallic with two idiomorphs, MAT1-1 and MAT1-2. Genomic sequencing of black (Elata clade) and yellow (Esculenta clade) morel species has led to the development of PCR primers designed to amplify genes from the two idiomorphs for rapid genotyping of isolates from these two clades. To evaluate the design and theoretical performance of these primers we performed a thorough bioinformatic investigation, including the detection of the MAT region in publicly available Morchella genomes and in-silico PCR analyses. All examined genomes, including those used for primer design, appeared to be heterothallic. This indicates an inherent fault in the original primer design which utilized a single Morchella genome, as the use of two genomes with complementary mating types would be required to design accurate primers for both idiomorphs. Furthermore, potential off-targets were identified for some of the previously published primer sets, but verification was challenging due to lack of adequate genomic information and detailed methodologies for primer design. Examinations of the black morel specific primer pairs (MAT11L/R and MAT22L/R) indicated the MAT22 primers would correctly target and amplify the MAT1-2 idiomorph, but the MAT11 primers appear to be capable of amplifying incorrect off-targets within the genome. The yellow morel primer pairs (EMAT1-1 L/R and EMAT1-2 L/R) appear to have reporting errors, as the published primer sequences are dissimilar with reported amplicon sequences and the EMAT1-2 primers appear to amplify the RNA polymerase II subunit ( RPB2 ) gene. The lack of the reference genome used in primer design and descriptive methodology made it challenging to fully assess the apparent issues with the primers for this clade. In conclusion, additional work is still required for the generation of reliable primers to investigate mating types in morels and to assess their performance on different clades and across multiple geographical regions.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2019

Additive Manufacturing for Designers: A Primer

Additive Manufacturing, also known as AM or 3D printing, is a class of manufacturing processes that create objects by shaping material layer by layer. Having demonstrated the ability to produce miraculously complex geometries, it is broadly claimed that AM will have endless applications as the technology improves. However, underneath the hype surrounding this technology is a world of nuance and constraints as well as highly strategic applications.Additive Manufacturing for Designers: A Primer, written by Dr. Amy Elliott from Oak Ridge National Laboratory and Dr. Cynthia K. Waters from North Carolina A&T State University discusses the topics needed for a holistic understanding of the many micro and macro components of the world of 3D printing.Additive Manufacturing for Designers: A Primer takes the reader on a journey beginning with important aspects of AM part design and process dependence, including resolution and tolerance issues of interest to any manufacturer. It also offers useful insights into how to build especially complex parts and avoid expensive mistakes during and after the manufacturing process is completed.

Elliott, Amy↗
DOE OSTI2016

Improved Bacterial 16S rRNA Gene (V4 and V4-5) and Fungal Internal Transcribed Spacer Marker Gene Primers for Microbial Community Surveys

We continue to uncover a wealth of information connecting microbes in important ways to human and environmental ecology. As our scientific knowledge and technical abilities improve, the tools used for microbiome surveys can be modified to improve the accuracy of our techniques, ensuring that we can continue to identify groundbreaking connections between microbes and the ecosystems they populate, from ice caps to the human body. It is important to confirm that modifications to these tools do not cause new, detrimental biases that would inhibit the field rather than continue to move it forward. We therefore demonstrated that two recently modified primer pairs that target taxonomically discriminatory regions of bacterial and fungal genomic DNA do not introduce new biases when used on a variety of sample types, from soil to human skin. This confirms the utility of these primers for maintaining currently recommended microbiome research techniques as the state of the art.

Walters, William↗
DOE OSTI2020

BigDNA

SAND2020-13696 M BigDNA takes a user input fast DNA sequence and outputs overlapping long PCR primers for Gibson Assembly. The software can design primers that remove gene products, insert new gene products, or completely circularize long DNAs. Sandia National Laboratories is a multimission laboratory managed and operated by National Technology & Engineering Solutions of Sandia, LLC, a wholly owned subsidiary of Honeywell International Inc., for the U.S. Department of Energy’s National Nuclear Security Administration under contract DE-NA0003525.

Porter, Kelley↗
DOE OSTI2021

Diagnostic Testing for COVID-19 Bridging Study for CDC EUA assays vs CDC Multiplex N1 FAM, N2 SUN, RNAse P ATTO 647 Assays

This report describes testing performed by LANL’s Biological Agent Testing Laboratory (BATL) to validate modifications to the CDC EUA 2019-Novel Coronavirus (2019-nCoV) Real-Time RT-PCR Diagnostic Panel (EUA-CDC-nCoV-IFU). BATL intends to implement the modification to increase thoughput for daily testing. BATL validated the original component, CDC designed primers and probes purchased from IDT (Cat # 10006770) and the new component, CDC assays designed with new Reporter dyes allowing the assays to be multiplexed, IDT (Cat # 10006830, 10006831, 10006823, 10006833, 10006834, 10007050, 10006836, 10006837, 10007062)

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Propidium Monoazide (PMAxx)-Recombinase Polymerase Amplification Exo (RPA Exo) Assay for Rapid Detection of Burkholderia cepacia Complex in Chlorhexidine Gluconate (CHX) and Benzalkonium Chloride (BZK) Solutions

Both sterile and non-sterile pharmaceutical products, which include antiseptics, have been recalled due to Burkholderia cepacia complex (BCC) contamination. Therefore, minimizing the frequency of outbreaks may be conducive to the development of a quick and sensitive approach that can distinguish between live and dead loads of BCC. We have assessed an exo probe-based recombinase polymerase amplification (RPA) with 10 µM propidium monoazide (PMAxx) for selective detection of live/dead BCC cells in various concentrations of antiseptics (i.e., chlorhexidine gluconate (CHX) and benzalkonium chloride (BZK) solutions) after 24 h. The optimized assay conducted using a set of primer–probes targeting gbpT was performed at 40 °C for 20 min and shows a detection limit of 10 pg/µL of genomic DNA from B. cenocepacia J2315, equivalent to 10 4 colony-forming units (CFU/mL). The specificity of a newly designed primer and probe was 80% (20 negatives out of 25). The readings for total cells (i.e., without PMAxx) from 200 µg/mL CHX using PMAxx-RPA exo assay was 310 relative fluorescence units (RFU), compared to 129 RFU with PMAxx (i.e., live cells). Furthermore, in 50–500 µg/mL BZK-treated cells, a difference in the detection rate was observed between the PMAxx-RPA exo assay in live cells (130.4–459.3 RFU) and total cells (207.82–684.5 RFU). This study shows that the PMAxx-RPA exo assay appears to be a valid tool for the simple, rapid and presumptive detection of live BCC cells in antiseptics, thereby ensuring the quality and safety of pharmaceutical products.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Populus VariantDB v3.2 facilitates CRISPR and functional genomics research

The success of CRISPR genome editing studies depends critically on the precision of guide RNA (gRNA) design. Sequence polymorphisms in outcrossing tree species pose design hazards that can render CRISPR genome editing ineffective. Despite recent advances in tree genome sequencing with haplotype resolution, sequence polymorphism information remains largely inaccessible to various functional genomics research efforts. The Populus VariantDB v3.2 addresses these challenges by providing a user-friendly search engine to query sequence polymorphisms of heterozygous genomes. The database accepts short sequences, such as gRNAs and primers, as input for searching against multiple poplar genomes, including hybrids, with customizable parameters. We provide examples to showcase the utilities of VariantDB in improving the precision of gRNA or primer design. The platform-agnostic nature of the probe search design makes Populus VariantDB v3.2 a versatile tool for the rapidly evolving CRISPR field and other sequence-sensitive functional genomics applications. The database schema is expandable and can accommodate additional tree genomes to broaden its user base.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2018

Association of Vampirovibrio chlorellavorus with decline and death of Chlorella sorokiniana in outdoor reactors

The outdoor ARID raceway was established for optimizing the cultivation of microalgae for biofuel production. During the summers of 2014 and 2015, discoloration was observed in cultures of Chlorella sorokiniana , which shifted from a vibrant green color to yellow, followed by cell clumping, decline in density, and rapid death, resulting in 40–60% reduced biomass production. To investigate, total DNA was purified from the raceway samples and subjected to polymerase chain reaction (PCR) amplification using degenerate primers that amplify the 16S rRNA gene of eubacteria. BLASTn analysis of the cloned amplicon sequences revealed the presence of the Gram-negative, predatory bacterium, Vampirovibrio chlorellavorus . Scanning electron microscopic examination showed an abundance of coccoid cells, 0.3–0.6 μm in diameter, some of which were attached to C. sorokiniana cells. PCR amplification indicated the presence of V. chlorellavorus in raceway vessels, water lines, connective tubing, and in early, scaled-up DOE1412 cultures used to inoculate the raceway. Based on PCR detection, the decontamination of the equipment and water line with "Wal-Clean" more effectively eliminated V. chlorellavorus and delayed the onset of attack, compared to the chlorine disinfectant, trichloromelamine (TCM). Total DNA was isolated from soil samples collected monthly from the nearby Rillito River during 2014–2015 and subjected to PCR amplification using primers designed to amplify the 16S rRNA and 18S rRNA gene of V. chlorellavorus and C. sorokiniana , respectively. Results indicated that V. chlorellavorus and Chlorella spp. were present in most of the riverbed samples nearly year round, suggesting a possible naturally occurring reservoir of the predatory bacterium.

59 BASIC BIOLOGICAL SCIENCES↗