Vasconcellea quercifolia (mamón del monte) es una especie nativa ampliamente distribuida en el centro-norte de Argentina. Constituye un potencial recurso forestal no maderero, sus frutos presentan ...excelentes propiedades organolépticas, su látex posee propiedades industriales y es fuente de resistencia a virus de importancia para el cultivo de papaya. El objetivo del estudio fue caracterizar la variabilidad genética en una colección de germoplasma de esta especie mediante el uso de marcadores moleculares. Se analizaron 50 genotipos procedentes de seis poblaciones mediante siete primers ISSR, los que fueron seleccionados en previa prueba de eficiencia y repetibilidad. Se calculó la distancia genética entre materiales mediante el coeficiente de Jaccard y se generó el dendrograma correspondiente mediante el algoritmo UPGMA. La proporción de fragmentos amplificados polimórficos fue baja y se observó reducida diversidad genética, incluso entre poblaciones geográficamente distantes. En el dendrograma, los genotipos no conformaron agrupamientos definidos y no se conglomeraron de modo concluyente de acuerdo a su procedencia. A pesar de ello, fue posible identificar poblaciones que resultaron claramente diferenciables entre sí para los marcadores empleados. Ante la baja diversidad genética de la colección se considera relevante ampliar el número de colectas desde sitios distantes a los analizados. Se remarca la necesidad de ampliar el número de primers o incorporar otras técnicas moleculares para poder profundizar en la caracterización de la variabilidad de este recurso fitogenético.
La hemocromatosis hereditaria (HH) es muy frecuente en la población caucásica. Se caracteriza por la presencia de depósitos de hierro, especialmente en hígado, y la cirrosis es su manifestación más ...importante. Existen 6 tipos de HH, siendo el gen HFE responsable de HH tipo I. La Porfiria Cutánea Tardía (PCT) Hereditaria o Adquirida se manifiesta clínicamente por factores desencadenantes, como la sobrecarga de hierro. Del estudio de las mutaciones C282Y y H63D en HFE en distintas poblaciones se observó que C282Y es más frecuente en el norte de Europa y de América que en la población mediterránea. Además, su frecuencia está aumentada en pacientes PCT. Nuestro objetivo fue estudiar la prevalencia de C282Y y H63D en pacientes con diagnóstico de HH (n= 163) y de PCT (n= 117) y compararlos con un grupo control (n= 93). Se amplificaron los exones 2 y 4 de HFE con primers específicos y los productos se secuenciaron automáticamente. El 58,1% del grupo PCT y el 65,6% del control no portaban estas mutaciones. En el grupo PCT, 31,6% portaba H63D (26,5% heterocigosis, 5,1% homocigosis), 6,8% C282Y (5,1% en heterocigosis, 1,7% en homocigosis) y 2,6% eran doble heterocigotas. En los HH, 61,9% portaba H63D (55,8% en heterocigosis, 6,1% en homocigosis), 31,3% C282Y (19,0% en heterocigosis, 12,3% en homocigosis) y 1,8% eran doble heterocigotas. Si bien se observó una mayor frecuencia de estas mutaciones en HH, no se encontraron diferencias significativas entre el grupo PCT y el control, indicando que en nuestro país el desencadenamiento de la PCT no estaría asociado a la presencia de estas mutaciones.
Con el fin de estudiar las interacciones en la red trófica en dos sistemas hortícolas se desarrolló un protocolo mediante la utilización de herramientas moleculares. La mosca blanca Trialeurodes ...vaporariorum y el pulgón Myzus persicae son plagas clave de hortalizas. El predador Tupiocoris cucurbitaceus y el parasitoide Encarsia formosa se encuentran asociados a moscas blancas, en tanto el predador Chrysoperla externa y el parasitoide Aphidius colemani son enemigos naturales de pulgones. El objetivo del trabajo fue desarrollar un protocolo de identificación molecular especie-específica para estudiar las relaciones tróficas entre las especies mencionadas. Cien hembras de T. cucurbitaceus (en inanición por 24 h) fueron alimentadas con ninfas de moscas blancas durante 3 h. Las hembras se fijaron a distintos tiempos de finalizada la alimentación. Por otra parte, ninfas de moscas blancas fueron expuestas a E. formosa durante 24 h. En los siguientes 12 días, 300 ninfas expuestas se fijaron diariamente para estimar el tiempo a partir del cual se puede detectar el parasitismo. En el segundo sistema se fijaron larvas de crisopas alimentadas con pulgones y se fijaron pulgones parasitados por A. colemani. Se secuenció una región de la COI en las distintas especies a partir de individuos en inanición. Se compararon las secuencias y se identificaron variantes distintivas de cada especie. En base a estas se diseñaron dos sets de primers especie-específicos y adecuados para realizar la detección simultánea de la presa, el predador y el parasitoide en ambos sistemas.
The deep sequencing of 16S rRNA genes amplified by universal primers has revolutionized our understanding of microbial communities by allowing the characterization of the diversity of the uncultured ...majority. However, some universal primers also amplify eukaryotic rRNA genes, leading to a decrease in the efficiency of sequencing of prokaryotic 16S rRNA genes with possible mischaracterization of the diversity in the microbial community. In this study, we compared 16S rRNA gene sequences from genome-sequenced strains and identified candidates for non-degenerate universal primers that could be used for the amplification of prokaryotic 16S rRNA genes. The 50 identified candidates were investigated to calculate their coverage for prokaryotic and eukaryotic rRNA genes, including those from uncultured taxa and eukaryotic organelles, and a novel universal primer set, 342F-806R, covering many prokaryotic, but not eukaryotic, rRNA genes was identified. This primer set was validated by the amplification of 16S rRNA genes from a soil metagenomic sample and subsequent pyrosequencing using the Roche 454 platform. The same sample was also used for pyrosequencing of the amplicons by employing a commonly used primer set, 338F-533R, and for shotgun metagenomic sequencing using the Illumina platform. Our comparison of the taxonomic compositions inferred by the three sequencing experiments indicated that the non-degenerate 342F-806R primer set can characterize the taxonomic composition of the microbial community without substantial bias, and is highly expected to be applicable to the analysis of a wide variety of microbial communities.
Directly labelling locus‐specific primers for microsatellite analysis is expensive and a common limitation to small‐budget molecular ecology projects. More cost‐effective end‐labelling of PCR ...products can be achieved through a three primer PCR approach, involving a fluorescently labelled universal primer in combination with modified locus‐specific primers with 5′ universal primer sequence tails. This technique has been widely used but has been limited largely due to a lack of available universal primers suitable for co‐amplifying large numbers of size overlapping loci and without requiring locus‐specific PCR conditions to be modified. In this study, we report a suite of four high‐performance universal primers that can be employed in a three primer PCR approach for efficient and cost‐effective fluorescent end‐labelling of PCR fragments. Amplification efficiency is maximized owing to high universal primer Tm values (approximately 60+ °C) that enhance primer versatility and enable higher annealing temperatures to be employed compared with commonly used universal primers such as M13. We demonstrate that these universal primers can be combined with multiple fluorophores to co‐amplify multiple loci efficiently via multiplex PCR. This method provides a level of multiplexing and PCR efficiency similar to microsatellite fluorescent detection assays using directly labelled primers while dramatically reducing project costs. Primer performance is tested using several alternative PCR strategies that involve both single and multiple fluorophores in single and multiplex PCR across a wide range of taxa.
Genome-walking is a molecular tool used to unveil uncharacterized DNA regions flanking a known DNA, which has been widely used in bioscience and related areas. This study developed a reliable and ...efficient PCR-based genome-walking approach, named as single primer site-specific nested PCR (SPN-PCR). A SPN-PCR set sequentially consists of three single-primer nested PCR amplifications. The primary relaxed thermal cycle promotes outmost nested site-specific primer (NSSP) to partially combine with numerous places on DNA template, synthesizing many single-stranded DNAs (ssDNA). Among them, the target ssDNA is exponentially amplified in the subsequent stringent cycles, as its 3′ part possesses the outmost NSSP complement; but a non-target ssDNA cannot be amplified, because it does not possess such a complement. Stringent secondary and tertiary PCRs also exclusively enrich this target DNA. Finally, the target DNA product becomes predominant. The feasibility of SPN-PCR was validated by genome-walking several selected genes from two divergent species.
•Each round of SPN-PCR is driven by a single NSSP.•Inner NSSP can partially anneal to an unknown region of interest at 60 °C.•Two rounds of SPN-PCRs suffice to give a positive genome-walking result.
Cutting of primers from reads is an important step of processing targeted amplicon-based next generation sequencing data. Existing tools are adapted for cutting of one or several primer/adapter ...sequences from reads and removing all of their occurrences. Also most of the existing tools use kmers and may cut only part of primers or primers with studied sequence of gene. Because of this, use of such programs leads to incorrect trimming, reduction of coverage, and increase in the number of false-positive and/or false-negative results. We have developed a new tool named cutPrimers for accurate cutting of any number of primers from reads. Using sequencing reads that were obtained during study of BRCA1/2 genes, we compared it with cutadapt, AlienTrimmer, and BBDuk. All of them trimmed reads in such a way that coverage of at least two amplicons decreased to unacceptable level (<30 reads) comparing with reads trimmed with cutPrimers. At the same time, Trimmomatic and AlienTrimmer cut all occurrences of primer sequences, so the length of the remaining reads was less than prospective.
Telomerase reverse transcribes short guanine (G)-rich DNA repeat sequences from its internal RNA template to maintain telomere length. G-rich telomere DNA repeats readily fold into G-quadruplex (GQ) ...structures in vitro, and the presence of GQ-prone sequences throughout the genome introduces challenges to replication in vivo. Using a combination of ensemble and single-molecule telomerase assays, we discovered that GQ folding of the nascent DNA product during processive addition of multiple telomere repeats modulates the kinetics of telomerase catalysis and dissociation. Telomerase reactions performed with telomere DNA primers of varying sequence or using GQ-stabilizing K⁺ versus GQ-destabilizing Li⁺ salts yielded changes in DNA product profiles consistent with formation of GQ structures within the telomerase–DNA complex. Addition of the telomerase processivity factor POT1–TPP1 altered the DNA product profile, but was not sufficient to recover full activity in the presence of Li⁺ cations. This result suggests GQ folding synergizes with POT1–TPP1 to support telomerase function. Single-molecule Förster resonance energy transfer experiments reveal complex DNA structural dynamics during real-time catalysis in the presence of K⁺ but not Li⁺, supporting the notion of nascent product folding within the active telomerase complex. To explain the observed distributions of telomere products, we globally fit telomerase time-series data to a kinetic model that converges to a set of rate constants describing each successive telomere repeat addition cycle. Our results highlight the potential influence of the intrinsic folding properties of telomere DNA during telomerase catalysis, and provide a detailed characterization of GQ modulation of polymerase function.
Since December 2019, the coronavirus disease 2019 (COVID-19) caused by a novel coronavirus SARS-CoV-2 has rapidly spread to almost every nation in the world. Soon after the pandemic was recognized by ...epidemiologists, a group of biologists comprising the ARTIC Network, has devised a multiplexed polymerase chain reaction (PCR) protocol and primer set for targeted whole-genome amplification of SARS-CoV-2. The ARTIC primer set amplifies 98 amplicons, which are separated only in two PCRs, across a nearly entire viral genome. The original primer set and protocol showed a fairly small amplification bias when clinical samples with relatively high viral loads were used. However, as sample's viral load become low, rapid decrease in abundances of several amplicons were seen. In this report, we will show that dimer formations between some primers are the major cause of coverage bias in the multiplex PCR. Based on this, we propose 12 alternative primers in total in the ARTIC primer set that were predicted to be involved in 14 primer interactions. The resulting primer set, version N1 (NIID-1), exhibits improved overall coverage compared to the ARTIC Network's original (V1) and modified (V3) primer set.
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