Search NASA⌕ Search

SEARCH · Search NASA

Results for “DNA Primers”

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.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 19 records
All sourcesNASA NTRSDOE OSTI
DOE OSTI2020

Mycobacterial DNA polymerase I: activities and crystal structures of the POL domain as apoenzyme and in complex with a DNA primer-template and of the full-length FEN/EXO–POL enzyme

Mycobacterial Pol1 is a bifunctional enzyme composed of an N-terminal DNA flap endonuclease/5' exonuclease domain (FEN/EXO) and a C-terminal DNA polymerase domain (POL). Here we document additional functions of Pol1: FEN activity on the flap RNA strand of an RNA:DNA hybrid and reverse transcriptase activity on a DNA-primed RNA template. We report crystal structures of the POL domain, as apoenzyme and as ternary complex with 3'-dideoxy-terminated DNA primer-template and dNTP. The thumb, palm, and fingers subdomains of POL form an extensive interface with the primer-template and the triphosphate of the incoming dNTP. Progression from an open conformation of the apoenzyme to a nearly closed conformation of the ternary complex entails a disordered-to-ordered transition of several segments of the thumb and fingers modules and an inward motion of the fingers subdomain—especially the O helix—to engage the primer-template and dNTP triphosphate. Distinctive structural features of mycobacterial Pol1 POL include a manganese binding site in the vestigial 3' exonuclease subdomain and a non-catalytic water-bridged magnesium complex at the protein-DNA interface. We report a crystal structure of the bifunctional FEN/EXO–POL apoenzyme that reveals the positions of two active site metals in the FEN/EXO domain.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Fine-scale evaluation of two standard 16S rRNA gene amplicon primer pairs for analysis of total prokaryotes and archaeal nitrifiers in differently managed soils

The advance of high-throughput molecular biology tools allows in-depth profiling of microbial communities in soils, which possess a high diversity of prokaryotic microorganisms. Amplicon-based sequencing of 16S rRNA genes is the most common approach to studying the richness and composition of soil prokaryotes. To reliably detect different taxonomic lineages of microorganisms in a single soil sample, an adequate pipeline including DNA isolation, primer selection, PCR amplification, library preparation, DNA sequencing, and bioinformatic post-processing is required. Besides DNA sequencing quality and depth, the selection of PCR primers and PCR amplification reactions arguably have the largest influence on the results. This study tested the performance and potential bias of two primer pairs, i.e., 515F (Parada)-806R (Apprill) and 515F (Parada)-926R (Quince) in the standard pipelines of 16S rRNA gene Illumina amplicon sequencing protocol developed by the Earth Microbiome Project (EMP), against shotgun metagenome-based 16S rRNA gene reads. The evaluation was conducted using five differently managed soils. We observed a higher richness of soil total prokaryotes by using reverse primer 806R compared to 926R, contradicting to in silico evaluation results. Both primer pairs revealed various degrees of taxon-specific bias compared to metagenome-derived 16S rRNA gene reads. Nonetheless, we found consistent patterns of microbial community variation associated with different land uses, irrespective of primers used. Total microbial communities, as well as ammonia oxidizing archaea (AOA), the predominant ammonia oxidizers in these soils, shifted along with increased soil pH due to agricultural management. In the unmanaged low pH plot abundance of AOA was dominated by the acid-tolerant NS-Gamma clade, whereas limed agricultural plots were dominated by neutral-alkaliphilic NS-Delta/NS-Alpha clades. This study stresses how primer selection influences community composition and highlights the importance of primer selection for comparative and integrative studies, and that conclusions must be drawn with caution if data from different sequencing pipelines are to be compared.

16S rRNA gene amplicon Illumina sequencing↗
DOE OSTI2022

High-throughput detection of T-DNA insertion sites for multiple transgenes in complex genomes

Abstract Background Genetic engineering of crop plants has been successful in transferring traits into elite lines beyond what can be achieved with breeding techniques. Introduction of transgenes originating from other species has conferred resistance to biotic and abiotic stresses, increased efficiency, and modified developmental programs. The next challenge is now to combine multiple transgenes into elite varieties via gene stacking to combine traits. Generating stable homozygous lines with multiple transgenes requires selection of segregating generations which is time consuming and labor intensive, especially if the crop is polyploid. Insertion site effects and transgene copy number are important metrics for commercialization and trait efficiency. Results We have developed a simple method to identify the sites of transgene insertions using T-DNA-specific primers and high-throughput sequencing that enables identification of multiple insertion sites in the T 1 generation of any crop transformed via Agrobacterium . We present an example using the allohexaploid oil-seed plant Camelina sativa to determine insertion site location of two transgenes. Conclusion This new methodology enables the early selection of desirable transgene location and copy number to generate homozygous lines within two generations.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Primer terminal ribonucleotide alters the active site dynamics of DNA polymerase η and reduces DNA synthesis fidelity

DNA polymerases catalyze DNA synthesis with high efficiency, which is essential for all life. Extensive kinetic and structural efforts have been executed in exploring mechanisms of DNA polymerases, surrounding their kinetic pathway, catalytic mechanisms, and factors that dictate polymerase fidelity. Recent time-resolved crystallography studies on DNA polymerase η (Pol η) and β have revealed essential transient events during the DNA synthesis reaction, such as mechanisms of primer deprotonation, separated roles of the three metal ions, and conformational changes that disfavor incorporation of the incorrect substrate. DNA-embedded ribonucleotides (rNs) are the most common lesion on DNA and a major threat to genome integrity. While kinetics of rN incorporation has been explored and structural studies have revealed that DNA polymerases have a steric gate that destabilizes ribonucleotide triphosphate binding, the mechanism of extension upon rN addition remains poorly characterized. Using steady-state kinetics, static and time-resolved X-ray crystallography with Pol η as a model system, we showed that the extra hydroxyl group on the primer terminus does alter the dynamics of the polymerase active site as well as the catalysis and fidelity of DNA synthesis. During rN extension, Pol η error incorporation efficiency increases significantly across different sequence contexts. Finally, our systematic structural studies suggest that the rN at the primer end improves primer alignment and reduces barriers in C2'-endo to C3'-endo sugar conformational change. Overall, our work provides further mechanistic insights into the effects of rN incorporation on DNA synthesis.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Structural basis of DNA synthesis opposite 8-oxoguanine by human PrimPol primase-polymerase

PrimPol is a human DNA polymerase-primase that localizes to mitochondria and nucleus and bypasses the major oxidative lesion 7,8-dihydro-8-oxoguanine (oxoG) via translesion synthesis, in mostly error-free manner. We present structures of PrimPol insertion complexes with a DNA template-primer and correct dCTP or erroneous dATP opposite the lesion, as well as extension complexes with C or A as a 3'–terminal primer base. We show that during the insertion of C and extension from it, the active site is unperturbed, reflecting the readiness of PrimPol to accommodate oxoG(anti). The misinsertion of A opposite oxoG(syn) also does not alter the active site, and is likely less favorable due to lower thermodynamic stability of the oxoG(syn)•A base-pair. During the extension step, oxoG(syn) induces an opening of its base-pair with A or misalignment of the 3'-A primer terminus. Together, the structures show how PrimPol accurately synthesizes DNA opposite oxidatively damaged DNA in human cells.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

PARA: A New Platform for the Rapid Assembly of gRNA Arrays for Multiplexed CRISPR Technologies

Multiplexed CRISPR technologies have great potential for pathway engineering and genome editing. However, their applications are constrained by complex, laborious and time-consuming cloning steps. In this research, we developed a novel method, PARA, which allows for the one-step assembly of multiple guide RNAs (gRNAs) into a CRISPR vector with up to 18 gRNAs. Here, we demonstrate that PARA is capable of the efficient assembly of transfer RNA/Csy4/ribozyme-based gRNA arrays. To aid in this process and to streamline vector construction, we developed a user-friendly PARAweb tool for designing PCR primers and component DNA parts and simulating assembled gRNA arrays and vector sequences.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Choice of 16S ribosomal RNA primers affects the microbiome analysis in chicken ceca

We evaluated the effect of applying different sets of 16S rRNA primers on bacterial composition, diversity, and predicted function in chicken ceca. Cecal contents from Ross 708 birds at 1, 3, and 5 weeks of age were collected for DNA isolation. Eight different primer pairs targeting different variable regions of the 16S rRNA gene were employed. DNA sequences were analyzed using open-source platform QIIME2 and the Greengenes database. PICRUSt2 was used to determine the predicted function of bacterial communities. Changes in bacterial relative abundance due to 16S primers were determined by GLMs. The average PCR amplicon size ranged from 315 bp (V3) to 769 bp (V4–V6). Alpha- and beta-diversity, taxonomic composition, and predicted functions were significantly affected by the primer choice. Beta diversity analysis based on Unweighted UniFrac distance matrix showed separation of microbiota with four different clusters of bacterial communities. Based on the alpha- and beta-diversity and taxonomic composition, variable regions V1–V3(1) and (2), and V3–V4 and V3–V5 were in most consensus. Our data strongly suggest that selection of particular sets of the 16S rRNA primers can impact microbiota analysis and interpretation of results in chicken as was shown previously for humans and other animal species.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Flap Endonuclease 1 Endonucleolytically Processes RNA to Resolve R-Loops through DNA Base Excision Repair

Flap endonuclease 1 (FEN1) is an essential enzyme that removes RNA primers and base lesions during DNA lagging strand maturation and long-patch base excision repair (BER). It plays a crucial role in maintaining genome stability and integrity. FEN1 is also implicated in RNA processing and biogenesis. A recent study from our group has shown that FEN1 is involved in trinucleotide repeat deletion by processing the RNA strand in R-loops through BER, further suggesting that the enzyme can modulate genome stability by facilitating the resolution of R-loops. However, it remains unknown how FEN1 can process RNA to resolve an R-loop. In this study, we examined the FEN1 cleavage activity on the RNA:DNA hybrid intermediates generated during DNA lagging strand processing and BER in R-loops. We found that both human and yeast FEN1 efficiently cleaved an RNA flap in the intermediates using its endonuclease activity. We further demonstrated that FEN1 was recruited to R-loops in normal human fibroblasts and senataxin-deficient (AOA2) fibroblasts, and its R-loop recruitment was significantly increased by oxidative DNA damage. We showed that FEN1 specifically employed its endonucleolytic cleavage activity to remove the RNA strand in an R-loop during BER. We found that FEN1 coordinated its DNA and RNA endonucleolytic cleavage activity with the 3'-5' exonuclease of APE1 to resolve the R-loop. Our results further suggest that FEN1 employed its unique tracking mechanism to endonucleolytically cleave the RNA strand in an R-loop by coordinating with other BER enzymes and cofactors during BER. Our study provides the first evidence that FEN1 endonucleolytic cleavage can result in the resolution of R-loops via the BER pathway, thereby maintaining genome integrity.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Structural basis for DNA proofreading

DNA polymerase (DNAP) can correct errors in DNA during replication by proofreading, a process critical for cell viability. However, the mechanism by which an erroneously incorporated base translocates from the polymerase to the exonuclease site and the corrected DNA terminus returns has remained elusive. Here, we present an ensemble of nine high-resolution structures representing human mitochondrial DNA polymerase Gamma, Polγ, captured during consecutive proofreading steps. The structures reveal key events, including mismatched base recognition, its dissociation from the polymerase site, forward translocation of DNAP, alterations in DNA trajectory, repositioning and refolding of elements for primer separation, DNAP backtracking, and displacement of the mismatched base into the exonuclease site. Altogether, our findings suggest a conserved ‘bolt-action’ mechanism of proofreading based on iterative cycles of DNAP translocation without dissociation from the DNA, facilitating primer transfer between catalytic sites. Functional assays and mutagenesis corroborate this mechanism, connecting pathogenic mutations to crucial structural elements in proofreading steps.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Using low volume eDNA methods to sample pelagic marine animal assemblages

Environmental DNA (eDNA) is an increasingly useful method for detecting pelagic animals in the ocean but typically requires large water volumes to sample diverse assemblages. Ship-based pelagic sampling programs that could implement eDNA methods generally have restrictive water budgets. Studies that quantify how eDNA methods perform on low water volumes in the ocean are limited, especially in deep-sea habitats with low animal biomass and poorly described species assemblages. Using 12S rRNA and COI gene primers, we quantified assemblages comprised of micronekton, coastal forage fishes, and zooplankton from low volume eDNA seawater samples (n = 436, 380–1800 mL) collected at depths of 0–2200 m in the southern California Current. We compared diversity in eDNA samples to concurrently collected pelagic trawl samples (n = 27), detecting a higher diversity of vertebrate and invertebrate groups in the eDNA samples. Differences in assemblage composition could be explained by variability in size-selectivity among methods and DNA primer suitability across taxonomic groups. The number of reads and amplicon sequences variants (ASVs) did not vary substantially among shallow (<200 m) and deep samples (>600 m), but the proportion of invertebrate ASVs that could be assigned a species-level identification decreased with sampling depth. Using hierarchical clustering, we resolved horizontal and vertical variability in marine animal assemblages from samples characterized by a relatively low diversity of ecologically important species. Low volume eDNA samples will quantify greater taxonomic diversity as reference libraries, especially for deep-dwelling invertebrate species, continue to expand.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Structural dissection of sequence recognition and catalytic mechanism of human LINE-1 endonuclease

Abstract Long interspersed nuclear element-1 (L1) is an autonomous non-LTR retrotransposon comprising ∼20% of the human genome. L1 self-propagation causes genomic instability and is strongly associated with aging, cancer and other diseases. The endonuclease domain of L1’s ORFp2 protein (L1-EN) initiates de novo L1 integration by nicking the consensus sequence 5′-TTTTT/AA-3′. In contrast, related nucleases including structurally conserved apurinic/apyrimidinic endonuclease 1 (APE1) are non-sequence specific. To investigate mechanisms underlying sequence recognition and catalysis by L1-EN, we solved crystal structures of L1-EN complexed with DNA substrates. This showed that conformational properties of the preferred sequence drive L1-EN’s sequence-specificity and catalysis. Unlike APE1, L1-EN does not bend the DNA helix, but rather causes ‘compression’ near the cleavage site. This provides multiple advantages for L1-EN’s role in retrotransposition including facilitating use of the nicked poly-T DNA strand as a primer for reverse transcription. We also observed two alternative conformations of the scissile bond phosphate, which allowed us to model distinct conformations for a nucleophilic attack and a transition state that are likely applicable to the entire family of nucleases. This work adds to our mechanistic understanding of L1-EN and related nucleases and should facilitate development of L1-EN inhibitors as potential anticancer and antiaging therapeutics.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Supplementary table, figures and DNA sequences of sorghum gene models SbiRTx430.01G455400 and SbiRTx.02G006600 that feature primers, gRNAs and indels created

In-context promoter bashing via genome editing is a route to identify and characterize critical regulatory regions that govern expression of genes of interest. The outcomes of in-context promoter bashing can be used to inform editing strategies to modulate the expression of selected gene models in a desired fashion. Here we employed in-context promoter bashing to characterize the proximal upstream regulatory regions of sorghum genes encoding phosphoenolpyruvate carboxykinase (Sb.PEPCK.BS, SbiTx430.01G455400) and alanine aminotransferase (SbiTx430.02G006600, SbAlaAT.BS), two proteins involved in the PCK C4 pathway. Characterized germinal edits within the targeted regions upstream of these two genes ranged in size from 138 bp up to 1790 bp. A 138 bp within the Sb.PEPCK.BS upstream region and a 1643 bp element within the Sb.AlaAT.BS upstream region were determined to be important for maintenance of transcription levels. No change in development or various physiological parameters was observed in characterized lineages carrying promoter edits. However, significant changes in seed reserves and a reduction in 100 seed weight were consistently observed, under both greenhouse and field environments, in plants carrying an edit in the promoter of Sb.PEPCK.BS gene were significantly reduced in transcript accumulation for this gene.

Quach, Truyen [Center for Plant Science Innovation↗
DOE OSTI2022

Highly efficient and simple SSPER and rrPCR approaches for the accurate site-directed mutagenesis of large and small plasmids

Advances are needed in the site-directed mutagenesis of large plasmids for protein structure-function studies, as current methods are often inefficient, complicated and time-consuming. Here two new methods are reported that overcome these difficulties, namely the single primer extension reaction (SSPER) strategy that reaches 100% efficiency and the reduce recycle PCR (rrPCR) method that is advantageous in generating single and pairwise combinations of mutations. Both methods are distinguished from current technologies by the addition of a step that easily removes the oligonucleotide primer(s) after the first reaction, thus allowing for the addition of a second reaction in chronological sequence to generate and isolate the appropriate DNA product with the site-directed mutation(s). High efficiency of the methods is demonstrated by generating single and paired combinations of the 11 site-directed mutations targeted on 5 different plasmid DNA templates ranging from 10 to 12 kb and 57–60% GC-content at a rate of 50–100%. Overall, the methods are demonstrated to be (i) highly accurate, allowing for screening of plasmids by DNA sequencing, (ii) streamlined to generate the mutations within a single day, (iii) cost-effective in requiring only two primers and two enzymes (DpnI and a proofreading DNA polymerase), (iv) straightforward in primer design, (v) applicable for both large and small plasmids, and (vi) easily implemented by entry level researchers.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

A Telomerase-Activated Magnetic Resonance Imaging Probe for Consecutively Monitoring Tumor Growth Kinetics and In Situ Screening Inhibitors

Telomerase has long been considered as a biomarker for cancer diagnosis and a therapeutic target for drug discovery. Detecting telomerase activity in vivo could provide more direct information of tumor progression and response to drug treatment, which, however, is hampered by the lack of an effective probe that can generate an output signal without a tissue penetration depth limit. Here in this study, using the principle of distance-dependent magnetic resonance tuning, we constructed a telomerase-activated magnetic resonance imaging probe (TAMP) by connecting superparamagnetic ferroferric oxide nanoparticles (SPFONs) and paramagnetic Gd-DOTA (Gd(III) 1,4,7,10- tetraazacyclododecane-1,4,7,10-tetraazacyclododecane-1,4,7,10-tet-raacetic acid) complexes via telomerase-responsive DNA motifs. Upon telomerase-catalyzed extension of the primer in TAMP, Gd-DOTA-conjugated oligonucleotides can be liberated from the surface of SPFONs through a DNA strand displacement reaction, restoring the T 1 signal of the Gd-DOTA for a direct readout of the telomerase activity. Here we show that, by tracking telomerase activity, this probe provides consistent monitoring of tumor growth kinetics during progression and in response to drug treatment and enables in situ screening of telomerase inhibitors in whole-animal models. This study provides an alternative toolkit for cancer diagnosis, treatment response assessment, and anticancer drug screening.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2022

The association between vaginal microbiota disorders and early missed abortion: A prospective study

Abstract Introduction The objective of this study was to explore the association between disordered vaginal microbiota and missed abortion to ascertain potential causes of missed abortion related to vaginal microbiology. Material and methods This study was a prospective cohort study with a comparison group (reference group). Vaginal secretions from the posterior fornix of women in the early stages of pregnancy were collected in the Xiangya Third Hospital of Central South University in Changsha, Hunan, China, from November 2018 to November 2019. A total of 54 cases of missed abortion (case group) and 50 cases of normal pregnancy requiring induced abortion (reference group) were analyzed. Bacterial DNA was extracted, hybridized with gene‐specific primers, and then detected using a bacterial chip. The composition and relative abundance of vaginal microbiota in the two groups were compared using α‐diversity analysis, β‐diversity analysis, and the linear discriminant analysis effect size method. Results The α‐diversity analysis showed that the Simpson index of the case group was lower than that of the reference group, whereas the Shannon index in the case group was higher. The relative abundance of Firmicutes in the case group (42.52%) was lower than in the reference group (51.03%, p < 0.05), as was the relative abundance of Lactobacillus (case group 16.51%, reference group 23.00%; p < 0.05). Interestingly, levels of Mycoplasma genitalium and Ureaplasma were lower in the case group ( p < 0.05). The relative abundance of Lactobacillus crispatus , Lactobacillus jensenii , and Lactobacillus gasseri was also significantly lower in the case group than in the reference group ( p < 0.05). The pathways enriched in the case group were predominantly related to metabolism, whereas the genetics‐related pathways were predominantly enriched in the reference group. Conclusions Bacteria are more diverse and unevenly distributed in patients with missed abortion. Decreases in the proportion of vaginal Lactobacillus and changes in Lactobacillus species in these patients may increase the chance of genital tract pathogenic bacterial infection. To our knowledge, our study was the first to observe that a decrease of Firmicutes levels in the vaginal microbiota might impair energy metabolism and have an association with missed abortion.

Sun, Dan↗
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 OSTI2022

In crystallo observation of three metal ion promoted DNA polymerase misincorporation

Error-free replication of DNA is essential for life. Despite the proofreading capability of several polymerases, intrinsic polymerase fidelity is in general much higher than what base-pairing energies can provide. Although researchers have investigated this long-standing question with kinetics, structural determination, and computational simulations, the structural factors that dictate polymerase fidelity are not fully resolved. Time-resolved crystallography has elucidated correct nucleotide incorporation and established a three-metal-ion-dependent catalytic mechanism for polymerases. Using X-ray time-resolved crystallography, we visualize the complete DNA misincorporation process catalyzed by DNA polymerase η. The resulting molecular snapshots suggest primer 3´-OH alignment mediated by A-site metal ion binding is the key step in substrate discrimination. Moreover, we observe that C-site metal ion binding preceded the nucleotidyl transfer reaction and demonstrate that the C-site metal ion is strictly required for misincorporation. Our results highlight the essential but separate roles of the three metal ions in DNA synthesis.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Catalytic DNA Polymerization Can Be Expedited by Active Product Release**

Abstract The sequence‐specific hybridization of DNA facilitates its use as a building block for designer nanoscale structures and reaction networks that perform computations. However, the strong binding energy of Watson–Crick base pairing that underlies this specificity also causes the DNA dehybridization rate to depend sensitively on sequence length and temperature. This strong dependency imposes stringent constraints on the design of multi‐step DNA reactions. Here we show how an ATP‐dependent helicase, Rep‐X, can drive specific dehybridization reactions at rates independent of sequence length, removing the constraints of equilibrium on DNA hybridization and dehybridization. To illustrate how this new capacity can speed up designed DNA reaction networks, we show that Rep‐X extends the range of conditions where the primer exchange reaction, which catalytically adds a domain provided by a hairpin template to a DNA substrate, proceeds rapidly.

Moerman, Pepijn G.↗