Short Stretches Of DNA Research Articles

Plasmids affect microindel mutations in Acinetobacter baylyi ADP1

Plasmids can impact the evolution of their hosts, e.g. due to carriage of mutagenic genes, through cross-talk with host genes or as result of SOS induction during transfer. Here we demonstrate that plasmids can affect the level of microindel mutations in the host genome. These mutations are driven by the production of single-stranded DNA molecules that invade replication forks at microhomologies and subsequently get integrated into the genome. Using the gammaproteobacterial model organism Acinetobacter baylyi, we show that carriage of broad host range plasmids from different incompatibility groups can cause microindel mutations directly or indirectly. The plasmid vector pQLICE belonging to the incompatibility group Q (IncQ) and replicating by a characteristic strand displacement mechanism can generate chromosomal microindel mutations directly with short stretches of DNA originating from pQLICE. In addition, results with the IncP plasmid vector pRK415 (theta replication mechanism) show that the presence of plasmids can increase microindel mutation frequencies indirectly (i.e., with chromosomal ectopic DNA), presumably through plasmid-chromosome interactions that lead to DNA damages. These results provide new mechanistic insights into the microindel mutation mechanism, suggesting that single-stranded DNA repair intermediates are the causing agents. By contrast, the IncN plasmid RN3 appears to suppress host microindel mutations. The suppression mechanism remains unknown. Other plasmids in this study (belonging to IncA/C2, IncW, pBBR incompatibility groups) confer ambiguous or no quantifiable mutagenic effects.

The translesion polymerase Pol Y1 is a constitutive component of the B. subtilis replication machinery.

Unrepaired DNA damage encountered by the cellular replication machinery can stall DNA replication, ultimately leading to cell death. In the DNA damage tolerance pathway translesion synthesis (TLS), replication stalling is alleviated by the recruitment of specialized polymerases to synthesize short stretches of DNA near a lesion. Although TLS promotes cell survival, most TLS polymerases are low-fidelity and must be tightly regulated to avoid harmful mutagenesis. The gram-negative bacterium Escherichia coli has served as the model organism for studies of the molecular mechanisms of bacterial TLS. However, it is poorly understood whether these same mechanisms apply to other bacteria. Here, we use in vivo single-molecule fluorescence microscopy to investigate the TLS polymerase Pol Y1 in the model gram-positive bacterium Bacillus subtilis. We find significant differences in the localization and dynamics of Pol Y1 in comparison to its E. coli homolog, Pol IV. Notably, Pol Y1 is constitutively enriched at or near sites of replication in the absence of DNA damage through interactions with the DnaN clamp; in contrast, Pol IV has been shown to be selectively enriched only upon replication stalling. These results suggest key differences in the roles and mechanisms of regulation of TLS polymerases across different bacterial species.

Performance of a 74-Microhaplotype Assay in Kinship Analyses.

Microhaplotypes (MHs) consisting of multiple SNPs and indels on short stretches of DNA are new and interesting loci for forensic genetic investigations. In this study, we analysed 74 previously defined MHs in two of the populations that our laboratory provides with forensic genetic services, Danes and Greenlanders. In addition to the 229 SNPs that originally made up the 74 MHs, 66 SNPs and 3 indels were identified in the two populations, and 45 of these variants were included in new definitions of the MHs, whereas 24 SNPs were considered rare and of little value for case work. The average effective number of alleles (Ae) was 3.2, 3.0, and 2.6 in Danes, West Greenlanders, and East Greenlanders, respectively. High levels of linkage disequilibrium were observed in East Greenlanders, which reflects the characteristics of this population that has a small size, and signs of admixture and substructure. Pairwise kinship simulations of full siblings, half-siblings, first cousins, and unrelated individuals were performed using allele frequencies from MHs, STRs and SNPs from Danish and Greenlandic populations. The MH panel outperformed the currently used STR and SNP marker sets and was able to differentiate siblings from unrelated individuals with a 0% false positive rate and a 1.1% false negative rate using an LR threshold of 10,000 in the Danish population. However, the panel was not able to differentiate half-siblings or first cousins from unrelated individuals. The results generated in this study will be used to implement MHs as investigative markers for relationship testing in our laboratory.

Yeast zinc cluster transcription factors involved in the switch from fermentation to respiration show interdependency for DNA binding revealing a novel type of DNA recognition.

In budding yeast, fermentation is the most important pathway for energy production. Under low-glucose conditions, ethanol is used for synthesis of this sugar requiring a shift to respiration. This process is controlled by the transcriptional regulators Cat8, Sip4, Rds2 and Ert1. We characterized Gsm1 (glucose starvation modulator 1), a paralog of Rds2 and Ert1. Genome-wide analysis showed that Gsm1 has a DNA binding profile highly similar to Rds2. Binding of Gsm1 and Rds2 is interdependent at the gluconeogenic gene FBP1. However, Rds2 is required for Gsm1 to bind at other promoters but not the reverse. Gsm1 and Rds2 also bind to DNA independently of each other. Western blot analysis revealed that Rds2 controls expression of Gsm1. In addition, we showed that the DNA binding domains of Gsm1 and Rds2 bind cooperatively in vitro to the FBP1 promoter. In contrast, at the HAP4 gene, Ert1 cooperates with Rds2 for DNA binding. Mutational analysis suggests that Gsm1/Rds2 and Ert1/Rds2 bind to short common DNA stretches, revealing a novel mode of binding for this class of factors. Two-point mutations in a HAP4 site convert it to a Gsm1 binding site. Thus, Rds2 controls binding of Gsm1 at many promoters by two different mechanisms: regulation of Gsm1 levels and increased DNA binding by formation of heterodimers.

The SWI/SNF nucleosome remodeler constrains enhancer activity during Drosophila wing development.

Chromatin remodeling is central to the dynamic changes in gene expression that drive cell fate determination. During development, the sets of enhancers that are accessible for use change globally as cells transition between stages. While transcription factors and nucleosome remodelers are known to work together to control enhancer accessibility, it is unclear how the short stretches of DNA that they individually unmask yield the kilobase-sized accessible regions characteristic of active enhancers. Here, we performed a genetic screen to investigate the role of nucleosome remodelers in control of dynamic enhancer activity. We find that the Drosophila Switch/Sucrose Non-Fermenting complex, BAP, is required for repression of a temporally dynamic enhancer, brdisc. Contrary to expectations, we find that the BAP-specific subunit Osa is dispensable for mediating changes in chromatin accessibility between the early and late stages of wing development. Instead, we find that Osa is required to constrain the levels of brdisc activity when the enhancer is normally active. Genome-wide profiling reveals that Osa directly binds brdisc as well as thousands of other developmentally dynamic regulatory sites, including multiple genes encoding components and targets of the Notch signaling pathway. Transgenic reporter analyses demonstrate that Osa is required for activation and for constraint of different sets of target enhancers in the same cells. Moreover, Osa loss results in hyperactivation of the Notch ligand Delta and development of ectopic sensory structures patterned by Notch signaling early in development. Together, these findings indicate that proper constraint of enhancer activity is necessary for regulation of dose-dependent developmental events.

Normalized Multipotential Redox Coding of DNA Bases for Determination of Total Nucleotide Composition.

The previously reported approach of orthogonal multipotential redox coding of all four DNA bases allowed only analysis of the relative nucleotide composition of short DNA stretches. Here, we present two methods for normalization of the electrochemical readout to facilitate the determination of the total nucleotide composition. The first method is based on the presence or absence of an internal standard of 7-deaza-2'-deoxyguanosine in a DNA primer. The exact composition of the DNA was elucidated upon two parallel analyses and the subtraction of the electrochemical signal intensities. The second approach took advantage of a 5'-viologen modified primer, with this fifth orthogonal redox label acting as a reference for signal normalization, thus allowing accurate electrochemical sequence analysis in a single read. Both approaches were tested using various sequences, and the voltammetric signals obtained were normalized using either the internal standard or the reference label and demonstrated to be in perfect agreement with the actual nucleotide composition, highlighting the potential for targeted DNA sequence analysis.

BarKeeper—a versatile web framework to assemble, analyse and manage DNA barcoding data and metadata

Abstract DNA barcoding aids in rapidly identifying specimens in various contexts by comparing short stretches of DNA to a reference database. Barcoding initiatives generate large reference databases of carefully curated high‐quality sequences to maximize identification success. BarKeeper provides a flexible tool for barcoding initiatives and in the context of phylogenetic studies to foster shared work on large datasets of raw sequence and associated metadata. It is free, open‐source and available as a set of Docker containers for easy setup. After setting it up once, all project members can use it independently of their operating system or location. BarKeeper offers features to collect and manage data and metadata about specimens, taxa and DNA sequences from Sanger sequencing and high‐throughput sequencing (HTS) technologies. It provides excellent flexibility by not being restricted to specific markers or taxon groups. Users can view and edit records and associated metadata while the app assists them by trimming and assembling reads. It automatically checks the quality of generated barcodes with a taxonomy‐based tool and offers a wide range of options for data analysis. Extensive search features allow querying the database for specific groups of records and saving the search results in the user's profile or downloading them in various file formats. BarKeeper combines multiple tools to aid barcoding projects in every step, from a reference taxa list to finished barcode sequences, thereby minimizing the number of laborious, potentially error‐prone manual steps and enabling efficient collaborative workflows.

Detection of Oxidatively Modified Base Lesion(s) in Defined DNA Sequences by FLARE Quantitative PCR.

Assessment of DNA base and strand damage can be determined using a quantitative PCR assay that is based on the concept that damage blocks the progression of a thermostable polymerase thus resulting in decreased amplification. However, some of the mutagenic DNA base lesions cause little or no distortion in Watson-Crick base pairing. One of the most abundant such lesion is 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo(d)Gua), although it affects the thermodynamic stability of the DNA, duplex 8-oxo(d)Gua does not inhibit DNA synthesis or arrest most of DNA or RNA polymerases during replication and transcription. When unrepaired, it is a pre-mutagenic base as it pairs with adenine in anti-syn conformation. Recent studies considered 8-oxo(d)Gua as an epigenetic-like mark and along with 8-oxoguanine DNA glycosylase1 (OGG1) and apurinic/apyrimidinic endonuclease1 (APE1) has roles in gene expression via nucleating transcription factor's promoter occupancy. Here, we introduce its identification through fragment length analysis with repair enzyme (FLARE)-coupled quantitative (q)-PCR. One of the strengths of the assay is that 8-oxo(d)Gua can be identified within short stretches of nuclear and mitochondrial DNA in ng quantities. Bellow we describe the benefits and limits of using FLARE qPCR to assess DNA damage in mammalian cells and provide a detailed protocol of the assay.

Unscheduled DNA Synthesis at Sites of Local UV-induced DNA Damage to Quantify Global Genome Nucleotide Excision Repair Activity in Human Cells.

Nucleotide excision repair (NER) removes a wide variety of structurally unrelated lesions from the genome, including UV-induced photolesions such as 6-4 pyrimidine-pyrimidone photoproducts (6-4PPs) and cyclobutane pyrimidine dimers (CPDs). NER removes lesions by excising a short stretch of single-stranded DNA containing the damaged DNA, leaving a single-stranded gap that is resynthesized in a process called unscheduled DNA synthesis (UDS). Measuring UDS after UV irradiation in non-dividing cells provides a measure of the overall NER activity, of which approximately 90% is carried out by the global genome repair (GGR) sub pathway. Here, we present a protocol for the microscopy-based analysis and quantification of UDS as a measurement for GGR activity. Following local UV-C irradiation, serum-starved human cells are supplemented with the thymidine analogue 5-ethynyl-2'-deoxyuridine (EdU), which is incorporated into repair patches following NER-dependent dual incision. The incorporated nucleotide analogue is coupled to a fluorophore using Click-iT chemistry, followed by immunodetection of CPD photolesions to simultaneously visualize both signals by fluorescence microscopy. Accompanying this protocol is a custom-built ImageJ plug-in to analyze and quantify UDS signals at sites of CPD-marked local damage. The local UDS assay enables an effective and sensitive fluorescence-based quantification of GGR activity in single cells with application in basic research to better understand the regulatory mechanism in NER, as well as in diagnostics to characterize fibroblasts from individuals with NER-deficiency disorder. Graphical abstract.

Prophages encode phage-defense systems with cognate self-immunity

SummaryTemperate phages are pervasive in bacterial genomes, existing as vertically inherited islands termed prophages. Prophages are vulnerable to predation of their host bacterium by exogenous phages. Here, we identify BstA, a family of prophage-encoded phage-defense proteins in diverse Gram-negative bacteria. BstA localizes to sites of exogenous phage DNA replication and mediates abortive infection, suppressing the competing phage epidemic. During lytic replication, the BstA-encoding prophage is not itself inhibited by BstA due to self-immunity conferred by the anti-BstA (aba) element, a short stretch of DNA within the bstA locus. Inhibition of phage replication by distinct BstA proteins from Salmonella, Klebsiella, and Escherichia prophages is generally interchangeable, but each possesses a cognate aba element. The specificity of the aba element ensures that immunity is exclusive to the replicating prophage, preventing exploitation by variant BstA-encoding phages. The BstA protein allows prophages to defend host cells against exogenous phage attack without sacrificing the ability to replicate lytically.

Carborane- or Metallacarborane-Linked Nucleotides for Redox Labeling. Orthogonal Multipotential Coding of all Four DNA Bases for Electrochemical Analysis and Sequencing.

We report a series of 2'-deoxyribonucleoside triphosphates bearing dicarba-nido-undecaborate ([C2B9H11]1-), [3,3'-iron-bis(1,2-dicarbollide)]- (FESAN, [Fe(C2B9H11)2]2-) or [3,3'-cobalt-bis(1,2-dicarbollide)]- (COSAN, [Co(C2B9H11)2]2-) groups prepared either through the Sonogashira cross-coupling or the CuAAC click reaction. The modified dNXTPs were substrates for KOD XL DNA polymerase in enzymatic synthesis of modified DNA through primer extension (PEX). The nido-carborane- and FESAN-modified nucleotides gave analytically useful oxidation signals in square-wave voltammetry and were used for redox labeling of DNA. The redox-modified DNA probes were prepared by PEX using tailed primers and were hybridized to electrode (gold or glassy carbon) containing capture oligonucleotides. The combination of nido-carborane- and FESAN-linked nucleotides with 7-ferrocenylethynyl-7-deaza-dATP and 7-deaza-dGTP allowed polymerase synthesis of DNA fully modified at all four nucleobases, and each of the redox labels gave four differentiable and ratiometric signals in voltammetry. Thus, the combination of these four redox labels constitutes the first fully orthogonal redox coding of all four canonical nucleobases, which can be used for determination of nucleobase composition of short DNA stretches in one simple PEX experiment with electrochemical readout.

Expression and purification of codon-optimized cre recombinase in E. coli

The presence of antibiotic resistance genes in genetically modified bacteria raises a regulatory concern in the production of therapeutic proteins and additionally reduces the number of plasmids available for propagation in a cell. Cre recombinase from bacteriophage P1, involved in Cre/loxP mechanism is one of the widely used systems for selectable marker gene removal. We have overexpressed codon-optimized cre gene in pColdIV and pET28a(+) vector systems and purified His6-Cre recombinase by immobilized metal affinity chromatography. N-terminal His6 tagged Cre recombinase obtained was approximately 26 fold purified and promoted the site-specific recombination of two loxP sites of linearized pLox2+ vector allowing the excision of a re-circularized plasmid and a short stretch of DNA containing the recombined loxP site. The results of the expression using two vectors, purification and activity assessment of His6 tagged Cre recombinase is presented here.

Branched unwinding mechanism of the Pif1 family of DNA helicases

Members of the Pif1 family of helicases function in multiple pathways that involve DNA synthesis: DNA replication across G-quadruplexes; break-induced replication; and processing of long flaps during Okazaki fragment maturation. Furthermore, Pif1 increases strand-displacement DNA synthesis by DNA polymerase δ and allows DNA replication across arrays of proteins tightly bound to DNA. This is a surprising feat since DNA rewinding or annealing activities limit the amount of single-stranded DNA product that Pif1 can generate, leading to an apparently poorly processive helicase. In this work, using single-molecule Förster resonance energy transfer approaches, we show that 2 members of the Pif1 family of helicases, Pif1 from Saccharomyces cerevisiae and Pfh1 from Schizosaccharomyces pombe, unwind double-stranded DNA by a branched mechanism with 2 modes of activity. In the dominant mode, only short stretches of DNA can be processively and repetitively opened, with reclosure of the DNA occurring by mechanisms other than strand-switching. In the other less frequent mode, longer stretches of DNA are unwound via a path that is separate from the one leading to repetitive unwinding. Analysis of the kinetic partitioning between the 2 different modes suggests that the branching point in the mechanism is established by conformational selection, controlled by the interaction of the helicase with the 3' nontranslocating strand. The data suggest that the dominant and repetitive mode of DNA opening of the helicase can be used to allow efficient DNA replication, with DNA synthesis on the nontranslocating strand rectifying the DNA unwinding activity.

Hotspots of Sequence Variability in Gut Microbial Genes Encoding Pro-Inflammatory Factors Revealed by Oligotyping.

The gut microbiota has been implicated in a number of normal and disease biological processes. Recent studies have identified a subset of gut bacterial genes as potentially involved in inflammatory processes. In this work, we explore the sequence variability for some of these bacterial genes using a combination of deep sequencing and oligotyping, a data analysis application that identifies mutational hotspots in short stretches of DNA. The genes for pks island, tcpC and usp, all harbored by certain strains of E. coli and all implicated in inflammation, were amplified by PCR directly from stool samples and subjected to deep amplicon sequencing. For comparison, the same genes were amplified from individual bacterial clones. The amplicons for pks island and tcpC from stool samples showed minimal levels of heterogeneity comparable with the individual clones. The amplicons for usp from stool samples, by contrast, revealed the presence of five distinct oligotypes in two different regions. Of these, the oligotype GT was found to be present in the control uropathogenic clinical isolate and also detected in stool samples from individuals with colorectal cancer (CRC). Mutational hotspots were mapped onto the USP protein, revealing possible substitutions around Leu110, Glu114, and Arg115 in the middle of the pyocin domain (Gln110, Gln114, and Thr115 in most healthy samples), and also Arg218 in the middle of the nuclease domain (His218 in the uropathogenic strain). All of these results suggest that a level of variability within bacterial pro-inflammatory genes could explain differences in bacterial virulence and phenotype.

Abstract 2300: Validation of highly sensitive ctDNA assays for ESR1 resistance mutations with the NGS TargetSelectorTM enrichment technology

Abstract Background: Acquired resistance to anti-hormonal therapy is a common reason for treatment failure in advanced and metastatic breast cancer (mBRC). Mutations in the ESR1 gene, encoding the estrogen receptor (ER) protein, are often associated with this drug resistance. Accurate and timely ESR1 mutation detection may enable early identification of progression and prediction of treatment failure. In contrast to using difficult and painful tissue biopsies, “liquid biopsy” offers a non-invasive and systemic approach to identify solid tumor mutations in peripheral blood by assessing circulating tumor DNA (ctDNA). Biocept has developed the highly sensitive TargetSelectorTM NGS assay to detect low frequency mutant alleles in ctDNA using a patented blocker that suppresses excess WT alleles, while still allowing amplification of tumor derived mutant sequences. Here, we focus on a five-codon ESR1 mutation hotspot that imparts tumor cell resistance to anti-hormonal therapy in mBRC. Methods: The TargetSelectorTM assay applies a specific blocker on a short stretch of target DNA (up to 13 bp). The ESR1 assay consists of two blockers in the region of codons 534-538 of the ESR1 gene. We validated the TargetSelectorTM assays by spiking low level g-block DNA fragments (IDT Inc.) carrying point mutations for each of the targeted ESR1 sites, into human placental DNA. Mutant sequences were enriched via PCR and mutations were subsequently confirmed on the Illumina MiSeq down to a limit of detection (LOD) of 0.01-0.05% minor allele frequency (MAF). For ctDNA testing, whole blood was collected in Biocept CEE-SureTM Blood Collection tubes, and total nucleic acid extraction from plasma was performed on the QIAsymphony. Results: In total, we tested >600 samples for ESR1 TargetSelectorTM ctDNA assays, with ESR1 WT as the background reference. The assay shows >99% analytical sensitivity (at 0.05% MAF) and >97% analytical specificity. Based on practical and theoretical estimates and using ddPCR as an orthogonal verification method, the ESR1assays demonstrate single mutant copy detection for most tested positions, and in all cases showed an LOD of at least 0.05% (5 mutant copies in a background of 10,000 WT copies). Samples tested from 20 healthy donors (40 tests in total) showed high clinical specificity, indicating the test is competent for use in a CLIA certified, CAP accredited laboratory setting. Conclusions: Biocept’s TargetSelectorTM ctDNA assays are validated both analytically and clinically, showing single mutant copy detection and LOD at 0.05% or better in a background of excess WT DNA. Implementation of the Illumina MiSeq platform into our TargetSelectorTM assays leads to higher sample throughput, greater sensitivity and a faster turnaround time than Sanger sequencing. These factors enable a sensitive and efficient test crucial for guiding treatment decisions and patient care. Citation Format: Shan-Fu Wu, Lyle J. Arnold, Jolly Shrivastava, Jason C. Poole. Validation of highly sensitive ctDNA assays for ESR1 resistance mutations with the NGS TargetSelectorTM enrichment technology [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2300.

DNA barcodes jump-start search for new species.

$180 million project aims to identify 2 million species.

Protein Lysine Acetylation Regulates the Choice of the Okazaki Fragment Maturation Pathway

Eukaryotic DNA replication machinery has evolved to accurately maintain the human genome through decades of life and across generational time. Mutations in the genome can initiate and promote human diseases, such as cancer and neurodegenerative disorders. During replication, lagging strand synthesis initiated by the error‐prone DNA polymerase alpha primes template DNA with a RNA/DNA initiator primer. This is further elongated by high‐fidelity DNA polymerase delta, creating short stretches of DNA, known as Okazaki fragments. Because millions of these fragments need to be created and accurately ligated per cell division, the risk of mutagenesis is higher in the lagging strand compared to the leading strand. Maturation of the Okazaki fragment necessitates the displacement of the initiator primer, to remove the RNA segment prior to ligation. Two redundant pathways have been proposed in this process, one, known as the short flap pathway, rapidly removes the RNA primer and allows ligation of the polymerase alpha synthesized DNA primer. The other pathway, known as the long flap pathway, promotes displacement of the entire initiator primer and the region is resynthesized by the high‐fidelity DNA polymerase delta. Many of the proteins involved in Okazaki fragment maturation pathway are post‐translationally modified by lysine acetylation. Biochemical studies analyzing enzymatic properties of in vitro acetylated Okazaki fragment proteins show alterations in protein‐DNA, protein‐protein interactions and enzyme function. Our preliminary analysis of in vitro acetylated recombinant Okazaki fragment processing (OFP) proteins show that lysine acetylation regulates enzyme activity in a manner consistent with promoting the long flap pathway. Lysine acetylation inhibits the function of the short flap pathway nuclease, flap endonuclease 1 (FEN1) and stimulates the functions of nearly all the proteins in the long flap pathway. Further, using reporter constructs, we showed that in mammalian cells, increasing cellular lysine acetylation correlated with use of the long flap pathway for repair processing. Our current model proposes that lysine acetylation of Okazaki fragment proteins regulates the choice of the long flap pathway, resulting in higher‐fidelity lagging strand synthesis.Support or Funding InformationThis work was supported by funding from National Institute of Health (GM098328) and IUPUI New Faculty Startup Award.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Abstract 4534: Validation of highly sensitive TargetSelectorTM ctDNA assays for EGFR, BRAF, and KRAS mutations

Abstract Background: Accurate detection of driver mutations in cancer patients is vital for targeted therapy. Compared to tissue biopsy, “liquid biopsy” offers a non-invasive and more systemic approach to identify tumor mutations by assessing circulating tumor DNA (ctDNA) released from tumor cells into peripheral blood. We have developed TargetSelectorTM Real-Time PCR based assays to detect low frequency mutant alleles in ctDNA. The TargetSelectorTM assay uses a proprietary blocker to suppress amplification of excess WT alleles released from normal cells, while allowing specific amplification of mutants. Here we focus on five important targets: EGFR (Del19, L858, and T790), BRAF (V600), and KRAS (G12/G13), which are relevant to lung cancer, melanoma, and colorectal cancer. Methods: The TargetSelectorTM assay applies a specific blocker to cover variants on a short stretch of target DNA (up to 10 bp for nucleotide variants). For example, one KRAS exon 2 blocker covers all variants on both G12 and G13 positions. The TargetSelectorTM assays were first validated with cancer cell line DNA carrying mutation targets on QuantStudio 5 Real-Time PCR instruments (QS5). Sanger Sequencing was subsequently performed to confirm the mutation. Analytical validation was conducted by 3 independent operators using 5 instruments across 5 days in our CAP/CLIA certified laboratory. For ctDNA testing, whole blood samples were collected in CEE-SureTM Blood Collection tubes and DNA extraction from plasma was performed on the QIAsymphony. Results: In total, we tested 3086 samples for EGFR, BRAF and KRAS TargetSelectorTM ctDNA assays, with EGFR WT assay as the background reference. All five ctDNA assays showed >99% analytical sensitivity and >99% analytical specificity. Based on practical and theoretical estimates, each ctDNA assay demonstrated single mutant copy detection sensitivity. In the presence of 14,000 copies of WT background, the sensitivity of our ctDNA assays are: EGFR Del19, 0.01%; EGFR L858R, 0.02%; EGFR T790M, 0.01%; BRAF V600E, 0.01%; KRAS G12C, 0.02%. The inter-assay and intra-assay analyses showed r2 >0.94, suggesting a consistent performance among operational variables. Samples tested from 20 healthy donors (100 tests in total) showed clinical specificity >99%. In the concordance study of 13 clinical samples (31 tests in total) between QS5 and ABI 7900HT platforms, TargetSelectorTM ctDNA assays with the QS5 identified the same plus additional mutations compared to the 7900HT. Conclusions: TargetSelectorTM ctDNA assays were validated both analytically and clinically, showing single mutant copy detection and sensitivity at 0.02% or better in a background of excess WT DNA. Implementation of the QS5 qPCR platform into our TargetSelectorTM ctDNA assays leads to a higher sensitivity and faster turnaround time. These factors enable sensitive and efficient testing crucial for guiding treatment decisions and patient care. Citation Format: Shan-Fu Wu, Timothy T. Lu, Anh Pham, Jeffrey Chen, Tony Daher, Errin Samuelsz, Manisha Patel, Veena M. Singh, Lyle J. Arnold, Jason C. Poole. Validation of highly sensitive TargetSelectorTM ctDNA assays for EGFR, BRAF, and KRAS mutations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4534.

Enzyme-Free Replication with Two or Four Bases.

All known forms of life encode their genetic information in a sequence of bases of a genetic polymer and produce copies through replication. How this process started before polymerase enzymes had evolved is unclear. Enzyme-free copying of short stretches of DNA or RNA has been demonstrated using activated nucleotides, but not replication. We have developed a method for enzyme-free replication. It involves extension with reversible termination, enzyme-free ligation, and strand capture. We monitored nucleotide incorporation for a full helical turn of DNA, during both a first and a second round of copying, by using mass spectrometry. With all four bases (A/C/G/T), an "error catastrophe" occurred, with the correct sequence being "overwhelmed" by incorrect ones. When only C and G were used, approximately half of the daughter strands had the mass of the correct sequence after 20 copying steps. We conclude that enzyme-free replication is more likely to be successful with just the two strongly pairing bases than with all four bases of the genetic alphabet.

Enhancer functions in three dimensions: beyond the flat world perspective.

Transcriptional enhancers constitute a subclass of regulatory elements that facilitate transcription. Such regions are generally organized by short stretches of DNA enriched in transcription factor-binding sites but also can include very large regions containing clusters of enhancers, termed super-enhancers. These regions increase the probability or the rate (or both) of transcription generally in cis and sometimes over very long distances by altering chromatin states and the activity of Pol II machinery at promoters. Although enhancers were discovered almost four decades ago, their inner workings remain enigmatic. One important opening into the underlying principle has been provided by observations that enhancers make physical contacts with their target promoters to facilitate the loading of the RNA polymerase complex. However, very little is known about how such chromatin loops are regulated and how they govern transcription in the three-dimensional context of the nuclear architecture. Here, we present current themes of how enhancers may boost gene expression in three dimensions and we identify currently unresolved key questions.