Damos las gracias a M. Meyer para el asesoramiento, la Academia Croata de Ciencias y Artes y la Academia de Berlín-Brandenburgo de Ciencias para el apoyo logístico y científico, y el Fondo de Innovación Presidencial de la Sociedad Max Planck para la ayuda financiera. JMG fue apoyado por una beca postdoctoral de NSF International (OISE-0754461). Las secuencias son depositados en la base de datos EBI nucleótidos con números de acceso: Neandertal 1 (Feldhofer 1) FM865407, Neandertal 2 (Feldhofer 2) FM865408, Sidrón 1253 FM865409, Vindija 33,25 FM865410, Mezmaiskaya un FM865411

Este método se utilizó en la investigación publicada en <a href="http://www.sciencemag.org/cgi/content/full/325/5938/318">Briggs et al., Science 325 (5938). Trescientas dieciocho-trescientos veintiuno (2009)</a> . Reactivos necesarios: <ul><li> AmpliTaq Gold ADN polimerasa </li><li> 10x GeneAmp PCR Buffer II </li><li> MgCl2 25 mM </li><li> Mezcla de dNTP, 25 mM de cada uno </li><li> BSA, 10 mg ml-1 en el agua </li><li> Biología molecular de agua de grado </li><li> EB tampón (suministrado con el kit de purificación MinElute PCR), 10 mM Tris-HCl, pH 8,5 </li><li> MinElute PCR kit de purificación </li><li> M-270 streptavidina Dynabeads </li><li> 2x encuadernación y lavado (BW) buffer (2 M NaCl, 10 mM Tris-Cl, 1 mM EDTA, pH 8,0, el 0,2% de Tween 20) </li><li> 1x encuadernación y lavado (BW) buffer (buffer 2x BindWash diluido en agua 2 veces) </li><li> Lave caliente (HW) buffer (2,5 mm de MgCl, Taq 1X buffer de oro, 0,1% Tween 20) </li></ul> Para información del fabricante acerca de la no-estándar de reactivos y equipo de la tabla más adelante. 1) Biblioteca de amplificación <ol><li> La plantilla de ADN que aquí hay una biblioteca de 454 preparados a partir de una fuente de copias de ADN de bajo número como se describe en (Rohland y Hofreiter, 2007a, 2007b) y (Maricic y Pääbo, 2009). Para ampliar la biblioteca de todo, preparar la siguiente mezcla de PCR: <table border="1" cellspacing="0" cellpadding="0"><tbody><tr><td> Reactivo </td><td> l por reacción </td></tr><tr><td> De agua </td><td> 45 </td></tr><tr><td> 10X Gene Amp PCR buffer II </td><td> 10 </td></tr><tr><td> MgCl 2 (25 mm) </td><td> 10 </td></tr><tr><td> BSA (10 mg / ml) </td><td> 2 </td></tr><tr><td> dNTPs (25 mM cada uno) </td><td> 1 </td></tr><tr><td> 454 emPCR FWD primer/10uM </td><td> 3 </td></tr><tr><td> 454 vto emPCR primer/10uM </td><td> 3 </td></tr><tr><td> AmpliTaq Gold ADN polimerasa (5 U / l) </td><td> 1 </td></tr><tr><td> 454 plantilla de ADN de la biblioteca </td><td> 25 </td></tr><tr><td> Total </td><td> 100 </td></tr></tbody></table></li><li> Utilice el siguiente programa de PCR en un termociclador Thermo por 14 ciclos de amplificación <ol><li> 95 ° C 12min </li><li> 95 ° C 30 </li><li> 60 ° C (temperatura deseada o recocido) 1 min </li><li> 72 ° C 1 min </li><li> Ir al 2 (x 13) </li><li> 72 ° C 5 minutos </li><li> 10 ° C ∞ </li></ol></li><li> Purificar la reacción de más de una columna de centrifugación Qiagen MinElute de acuerdo a las instrucciones del fabricante. Eluyen en 50 l EB buffer. </li><li> Cuantificar el producto purificado amplificados, así como una parte alícuota de la biblioteca sin amplificar con PCR cuantitativa (Meyer et al., 2007). Si el producto amplificado tiene más de 1 X 10 12 copias por ul, reducir la cantidad de plantilla en el paso de extensión del cebador siguiente para asegurar que no más de 2 X 10 12 ejemplares se añaden a la reacción de extensión de los iniciadores. </li></ol> 2) Primer Extensión <ol><li> Prepare una mezcla maestra para el número requerido de las reacciones. <table border="0" cellspacing="0" cellpadding="0"><tbody><tr><td> Reactivo </td><td> l por reacción </td></tr><tr><td> De agua </td><td> 45 </td></tr><tr><td> 10X Gene Amp PCR buffer II </td><td> 10 </td></tr><tr><td> MgCl 2 (25 mm) </td><td> 10 </td></tr><tr><td> BSA (10 mg / ml) </td><td> 2 </td></tr><tr><td> dNTPs (25 mM cada uno) </td><td> 1 </td></tr><tr><td> 454 emPCR FWD primer/10uM </td><td> 3 </td></tr><tr><td> 454 vto emPCR primer/10uM </td><td> 3 </td></tr><tr><td> AmpliTaq Gold ADN polimerasa (5 U / l) </td><td> 1 </td></tr><tr><td> 454 plantilla de ADN de la biblioteca </td><td> 25 </td></tr><tr><td> Total </td><td> 100 </td></tr></tbody></table></li><li> Aplicar el siguiente programa en un termociclador para la reacción de extensión del cebador único: <ol><li> 95 ° C 12min </li><li> 60 ° C (o la imprimación PEC recocido temp si es diferente) 1 min </li><li> 72 ° C 5 min </li><li> 72 ° C para siempre! Paso crítico para mantener la reacción después de la extensión a 72 ° C. Luego directamente pipeta 150ul PBI o tampón PB de purificación QIAGEN MinElute en los tubos antes de sacarlo del bloque de calor. Esto es importante para evitar la no específica primer recocido y captura ya que la mezcla se enfríe. </li></ol></li><li> Purificar la reacción de más de una columna de centrifugación Qiagen MinElute, de acuerdo con las instrucciones del fabricante. Eluyen en 50 l EB. </li></ol> 3) Extensión de la captura del producto <ol><li> Resuspender solución madre de M-270 granos por agitación. Saque 25 l de suspensión de cuentas por muestra. Lave las cuentas dos veces con 500 ul 2x BW buffer y resuspender en 25 l de amortiguación 2xBW por muestra. </li><li> Añadir 25 l eluido a partir del paso desde 2,3 hasta 25 l de suspensión de cuentas desde el paso 3.1. Mezclar a continuación, gire de un5 min a temperatura ambiente. Recomendado: mantener restante 5 ul de que el efluente de la etapa 2.3 para la cuantificación de qPCR. </li><li> La transferencia de toda la mezcla a un tubo de 1,5 ml frescos (esto ayuda a reducir el arrastre de fragmentos de la biblioteca no objetivo). Pellet el sobrenadante con el colector de partículas magnéticas (MPC) y descartar el sobrenadante. </li><li> Lavar 5 veces con buffer de 500 l 1xBW (lavados ayudan a eliminar como telón de fondo tanto como sea posible). Después de la última etapa de lavado, centrifugar brevemente y eliminar los últimos restos de sobrenadante. </li><li> Agregar 500μl 1x Wash caliente (HW) de amortiguación. Agitar durante 2 min a 65 ° C (5C o por encima de la Tm PEC primer) en un bloque térmico. Eliminar el sobrenadante rápidamente después de esto, para minimizar el enfriamiento. (Este paso es para eliminar los fragmentos de fondo todavía se asocia con los primers PEC no, pero en realidad extendida durante la etapa de extensión). Eliminar los últimos restos de sobrenadante. </li><li> Resuspender el precipitado en 30 bolas buffer EB l. La transferencia de toda la mezcla a un tubo de 1,5 ml frescos (esto ayuda a reducir el arrastre de fragmentos de la biblioteca no objetivo). Se incuba a 95 ° C durante 3 minutos en un termociclador para la elución. Lugar en el MPC y eliminar el sobrenadante, teniendo cuidado de dejar las cuentas atrás. </li></ol> 4) La captura de amplificación de productos <ol><li> Prepare una mezcla maestra de PCR para el número requerido de muestras. <table border="1" cellspacing="0" cellpadding="0"><tbody><tr><td> Reactivo </td><td> l por reacción </td></tr><tr><td> De agua </td><td> 45 </td></tr><tr><td> 10X Gene Amp PCR buffer II </td><td> 10 </td></tr><tr><td> MgCl 2 (25 mm) </td><td> 10 </td></tr><tr><td> BSA (10 mg / ml) </td><td> 2 </td></tr><tr><td> dNTPs (25 mM cada uno) </td><td> 1 </td></tr><tr><td> 454 emPCR FWD primer/10uM </td><td> 3 </td></tr><tr><td> 454 vto emPCR primer/10uM </td><td> 3 </td></tr><tr><td> AmpliTaq Gold ADN polimerasa (5 U / l) </td><td> 1 </td></tr><tr><td> Productos de captura eluye </td><td> 25 </td></tr><tr><td> Total </td><td> 100 </td></tr></tbody></table></li><li> Utilice el siguiente programa de PCR en un termociclador Thermo por 14 ciclos de amplificación: <ol><li> 95 ° C 12min </li><li> 95 ° C 30 </li><li> 60 ° C (temperatura deseada o recocido) 1 min </li><li> 72 ° C 1 min </li><li> Ir al 2 (x 13) </li><li> 72 ° C 5 minutos </li><li> 10 ° C ∞ </li></ol></li><li> Purificar la reacción sobre una columna Qiagen MinElute giro de sílice de acuerdo a las instrucciones del fabricante. Eluyen en 50 tampón EB l. El producto puede ser ahora utilizado, ya sea para una segunda ronda de la captura, a partir del paso 2.1, o introducirse directamente en el protocolo 454 emulsión PCR, para la secuenciación. </li></ol> Los resultados representativos: Es muy recomendable cuando se inicia con el protocolo de PEC para llevar a cabo una primera reacción de captura en una pequeña cantidad de una &quot;secuencia positiva objetivo de control (por ejemplo, a ~ 100 pb producto de PCR - un picogramo es bastante facilidad) se mezcla con la normal recomendado cantidad de amplificación 454 plantilla de la biblioteca (véase el paso 2.1), y la captura se realiza con una sola cartilla PEC diseñado para capturar el producto de control positivo. Si esta reacción se realiza el control, tanto positivas como qPCR con un control específico y 454 específica del adaptador de pares de cebadores se pueden utilizar para cuantificar la cantidad de &quot;objetivo de control&quot; frente a fondo en la reacción de extensión del cebador purificada (1,3), primer producto de extensión (2,3 ) y el producto se eluyó la captura de grano (3,6). De esta manera, tanto la eficacia de la eliminación del fondo (la &quot;especificidad&quot;) y la eficiencia de recuperación de destino (la &quot;sensibilidad&quot;) en sus condiciones experimentales se puede medir directamente. El éxito de protocolo PEC normalmente tiene las siguientes propiedades - Sensibilidad (medido por la cantidad de objetivo de control mantenido a través del protocolo): Monto total de la meta de control de producto eluido captura de grano (3,6) = ~ 20.1% del total de purificar la reacción de extensión del cebador (2,3). Fondo (medida por 454 el primer par emPCR): Monto total del fondo en el producto eluido captura de grano (3,6) &lt;0,01% del importe total en la reacción de extensión del cebador purificada (2,3). Si hay menos de 1% de la meta que queda en el producto final, el paso de extensión del cebador puede haber tenido éxito y debe ser investigada. Si no es mucho más que el 0,01% del fondo que queda en los productos finales, los pasos de lavado puede haber sido mal realizado y debe ser investigada.

<ol>
	<li>Briggs, A. W., Good, J. M., Green, R. E., Krause, J., Maricic, T., Stenzel, U., Lalueza-Fox, C., Rudan, P., Brajkovic, D., Kucan, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+retrieval+and+analysis+of+five+Neandertal+mtDNA+genomes.">Targeted retrieval and analysis of five Neandertal mtDNA genomes.</a> Science. 325 (5938), 318-321 (2009).</li><li>Rohland, N., Hofreiter, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+and+optimization+of+ancient+DNA+extraction.">Comparison and optimization of ancient DNA extraction.</a> Biotechniques. 42 (3), 343-352 (2007).</li><li>Rohland, N., Hofreiter, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ancient+DNA+extraction+from+bones+and+teeth.">Ancient DNA extraction from bones and teeth.</a> Nat Protoc. 2 (7), 1756-1762 (2007).</li><li>Maricic, T., P&#228;&#228;bo, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimization+of+454+sequencing+library+preparation+from+small+amounts+of+DNA+permits+sequence+determination+of+both+DNA+strands.">Optimization of 454 sequencing library preparation from small amounts of DNA permits sequence determination of both DNA strands.</a> Biotechniques. 46 (1), 51-57 (2009).</li><li>Meyer, M., AW, B. r. i. g. g. s., Maricic, T., Hober, B., Hoffner, B., Krause, J., Weihmann, A., Paabo, S., Hofreiter, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=From+micrograms+to+picograms:+quantitative+PCR+reduces+the+material+demands+of+high-throughput+sequencing.">From micrograms to picograms: quantitative PCR reduces the material demands of high-throughput sequencing.</a> Nucleic Acids Res. 36 (1), e5-e5 (2007).</li></ol>

The authors have nothing to disclose.

El método PEC es sencilla, rápida, sensible y específica. Por lo tanto, los autores prevén múltiples aplicaciones fuera de ADN antiguo, como la captura de pequeños fragmentos de ARN de una biblioteca de ARN, el interrogatorio de la variación estructural en una muestra colectiva o la captura de 16S (o loci otros) la diversidad de una muestra de metagenómica. Un punto a mencionar es que la sensibilidad de la captura se convierte en menor medida que el número de PEC cebadores en una reacción aumenta múltiple captura. PEC no es por tanto, ideal para la captura de las regiones de la captura de gran tamaño (por ejemplo, una megabase o más), pero está muy bien adaptado para la captura de las regiones objetivo pequeño o incluso posiciones de SNP de muchas personas de una manera rápida.

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		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
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<td>AmpliTaq Gold polymerase with GeneAmp PCR buffer II and MgCl2</td>
<td>&nbsp;</td>
<td>Applied Biosystems</td>
<td>N8080241</td>
<td>&nbsp;</td>
<tr>
<td>QIAGEN MinElute PCR purification kit</td>
<td>&nbsp;</td>
<td>Qiagen</td>
<td>28004</td>
<td>&nbsp;</td>
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<td>M-270 streptavidin beads</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>653.05</td>
<td>&nbsp;</td>
<tr>
<td>Tween-20</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>P2287</td>
<td>&nbsp;</td>
<tr>
<td>DynaMag-2 magnetic particle separator</td>
<td>&nbsp;</td>
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primer extension capture: targeted sequence retrieval from heavily degraded dna sources

Se presenta un método de recuperación dirigidos secuencia de ADN de fuentes de ADN que están muy degradados y contaminados con ADN microbiano, como es típico de huesos antiguos. El método reduce en gran medida la destrucción de la muestra y las demandas en relación con la secuenciación directa de PCR o métodos de secuenciación shotgun. Hemos utilizado este método para reconstruir la completa ADN mitocondrial (ADNmt) de los cinco genomas neandertales de toda su área geográfica. La diversidad genética mitocondrial de los neandertales tarde fue aproximadamente tres veces menor que la de los humanos modernos contemporáneos. Junto con el análisis de la evolución de proteínas ADN mitocondrial, estos datos sugieren que el tamaño de largo plazo efectivo de la población de neandertales era más pequeño que el de los humanos modernos y los grandes simios existentes.

Watch this Scientific Journal Video about Primer Extension Capture: Targeted Sequence Retrieval from Heavily Degraded DNA Sources at JoVE.com

Primer Extension Capture: Targeted Sequence Retrieval from Heavily Degraded DNA Sources

Se presenta un método de recuperación de antiguos dirigidos secuencia de ADN, que se utiliza para reconstruir el genoma mitocondrial completo de cinco individuos neandertales. La comparación de estas secuencias con los seres humanos actuales sugieren que los neandertales tenían una baja capacidad a largo plazo efectivo de la población.

Primer captura Extensión: Secuencia de recuperación selectiva de las fuentes de ADN muy degradado

We present a method of targeted DNA sequence retrieval from DNA sources which are heavily degraded and contaminated with microbial DNA, as is typical of ...

primer-extension-capture-targeted-sequence-retrieval-from-heavily

Research

JoVE Journal

Biology

20.0K vistas.  Max-Planck Institute for Evolutionary Anthropology, Leipzig. Se presenta un método de recuperación de antiguos dirigidos secuencia de ADN, que se utiliza para reconstruir el genoma mitocondrial completo de cinco individuos neandertales. La comparación de estas secuencias con los seres humanos actuales sugieren que los neandertales tenían una baja capacidad a largo plazo efectivo de la población.

Video: Primer Extension Capture: Targeted Sequence Retrieval from Heavily Degraded DNA Sources

Retrieval of Mouse Oocytes

To date, only a few studies have reported successful manipulations of Peromyscus embryogenesis or reproductive biology.  Together with the Peromyscus Genetic Stock Center (http://stkctr.biol.sc.edu), we are characterizing the salient differences needed to develop this system.  A primary goal has been to optimize oocyte/early embryo retrieval.

This protocol illustrates the technique for extracting oocytes or early-stage fertilized embryos from the oviduct of mice. The ability to identify the infindibulum and insert a blunt end needle into it is essential to correctly performing the procedure.

Recuperación de los ovocitos de ratón

To date, only a few studies have reported successful manipulations of Peromyscus embryogenesis or reproductive biology.  Together with the Peromyscus ...

Investigación

Biología

Proboscis Extension Response (PER) Assay in Drosophila

Proboscis extension response (PER) is a taste behavior assay that has been used in flies as well as in honeybees.
On the surface of the fly's mouth (labellum), there are hair-like structures called sensilla which houses taste neurons. When an attractive substance makes contact to the labellum, the fly extends its proboscis to consume the material. Proboscis Extension Response (PER) assay measures this taste behavior response, and it is a useful method to learn about food preferences in a single fly. Solutions of various sugars, such as sucrose, glucose and fructose, are very attractive to the fly. The effect of aversive substances can also be tested as reduction of PER when mixed in a sweet solution.Despite the simplicity of the basic procedure, there are many things that can prevent it from working. One of the factors that requires attention is the fly's responsive state. The required starvation time to bring the fly to the proper responsive state varies drastically from 36 to 72 hours. We established a series of controls to evaluate the fly's state and which allows screening out of non-responsive or hyper-responsive individual animals. Another important factor is the impact level and the position of the contact to the labellum, which would be difficult to describe by words. This video presentation demonstrates all these together with several other improvements that would increase the reproducibility of this method.

Proboscis Extension Response PER Assay in Drosophila

Proboscis extension response or PER is a taste behavior assay that has been used in flies as well as in honeybees. When the proboscis makes contact with an attractive substance, the fly extends its proboscis to consume the substance. Solutions of various sugars are very attractive to the fly.

Extensión probóscide Respuesta (PER) Ensayo en Drosophila

Proboscis extension response (PER) is a taste behavior assay that has been used in flies as well as in honeybees.
On the surface of the fly's mouth ...

Isolation of Genomic DNA from Mouse Tails

El aislamiento de ADN genómico de colas de ratón

Primer-Free Aptamer Selection Using A Random DNA Library

Aptamers are highly structured oligonucleotides (DNA or RNA) that can bind to targets with affinities comparable to antibodies 1. They are identified through an in vitro selection process called Systematic Evolution of Ligands by EXponential enrichment (SELEX) to recognize a wide variety of targets, from small molecules to proteins and other macromolecules 2-4. Aptamers have properties that are well suited for in vivo diagnostic and/or therapeutic applications: Besides good specificity and affinity, they are easily synthesized, survive more rigorous processing conditions, they are poorly immunogenic, and their relatively small size can result in facile penetration of tissues.
Aptamers that are identified through the standard SELEX process usually comprise ~80 nucleotides (nt), since they are typically selected from nucleic acid libraries with ~40 nt long randomized regions plus fixed primer sites of ~20 nt on each side. The fixed primer sequences thus can comprise nearly ~50% of the library sequences, and therefore may positively or negatively compromise identification of aptamers in the selection process 3, although bioinformatics approaches suggest that the fixed sequences do not contribute significantly to aptamer structure after selection 5. To address these potential problems, primer sequences have been blocked by complementary oligonucleotides or switched to different sequences midway during the rounds of SELEX 6, or they have been trimmed to 6-9 nt 7, 8. Wen and Gray 9 designed a primer-free genomic SELEX method, in which the primer sequences were completely removed from the library before selection and were then regenerated to allow amplification of the selected genomic fragments. However, to employ the technique, a unique genomic library has to be constructed, which possesses limited diversity, and regeneration after rounds of selection relies on a linear reamplification step. Alternatively, efforts to circumvent problems caused by fixed primer sequences using high efficiency partitioning are met with problems regarding PCR amplification 10.
We have developed a primer-free (PF) selection method that significantly simplifies SELEX procedures and effectively eliminates primer-interference problems 11, 12. The protocols work in a straightforward manner. The central random region of the library is purified without extraneous flanking sequences and is bound to a suitable target (for example to a purified protein or complex mixtures such as cell lines). Then the bound sequences are obtained, reunited with flanking sequences, and re-amplified to generate selected sub-libraries. As an example, here we selected aptamers to S100B, a protein marker for melanoma. Binding assays showed Kd s in the 10-7 - 10-8 M range after a few rounds of selection, and we demonstrate that the aptamers function effectively in a sandwich binding format.

SELEX protocols comprise multiple rounds of selection, each of which require regeneration of bound ligands, which in turn require fixed primer sequences flanking the random library regions. These fixed primer sequences can interfere with the selection process (false positives and negatives). Here we present a primer-free protocol.

Primer Libre Selección aptámero utilizar una biblioteca de ADN al azar

Aptamers are highly structured oligonucleotides (DNA or RNA) that can bind to targets with affinities comparable to antibodies 1. They are identified ...

Genomic MRI - a Public Resource for Studying Sequence Patterns within Genomic DNA

Non-coding genomic regions in complex eukaryotes, including intergenic areas, introns, and untranslated segments of exons, are profoundly non-random in their nucleotide composition and consist of a complex mosaic of sequence patterns. These patterns include so-called Mid-Range Inhomogeneity (MRI) regions -- sequences 30-10000 nucleotides in length that are enriched by a particular base or combination of bases (e.g. (G+T)-rich, purine-rich, etc.). MRI regions are associated with unusual (non-B-form) DNA structures that are often involved in regulation of gene expression, recombination, and other genetic processes (Fedorova &amp; Fedorov 2010). The existence of a strong fixation bias within MRI regions against mutations that tend to reduce their sequence inhomogeneity additionally supports the functionality and importance of these genomic sequences (Prakash et al. 2009).
Here we demonstrate a freely available Internet resource -- the Genomic MRI program package -- designed for computational analysis of genomic sequences in order to find and characterize various MRI patterns within them (Bechtel et al. 2008). This package also allows generation of randomized sequences with various properties and level of correspondence to the natural input DNA sequences. The main goal of this resource is to facilitate examination of vast regions of non-coding DNA that are still scarcely investigated and await thorough exploration and recognition.

We present a public computational web site for the analysis of genomic sequences. It detects DNA sequence patterns with various non-random nucleotide compositions. This resource also generates randomized sequences with diverse levels of complexity.

Genómica resonancia magnética - un recurso público para el estudio de patrones de secuencia en el ADN genómico

Non-coding genomic regions in complex eukaryotes, including intergenic areas, introns, and untranslated segments of exons, are profoundly non-random in ...

Selective Capture of 5-hydroxymethylcytosine from Genomic DNA

5-methylcytosine (5-mC) constitutes ~2-8% of the total cytosines in human genomic DNA and impacts a broad range of biological functions, including gene expression, maintenance of genome integrity, parental imprinting, X-chromosome inactivation, regulation of development, aging, and cancer1. Recently, the presence of an oxidized 5-mC, 5-hydroxymethylcytosine (5-hmC), was discovered in mammalian cells, in particular in embryonic stem (ES) cells and neuronal cells2-4. 5-hmC is generated by oxidation of 5-mC catalyzed by TET family iron (II)/&alpha;-ketoglutarate-dependent dioxygenases2, 3. 5-hmC is proposed to be involved in the maintenance of embryonic stem (mES) cell, normal hematopoiesis and malignancies, and zygote development2, 5-10. To better understand the function of 5-hmC, a reliable and straightforward sequencing system is essential. Traditional bisulfite sequencing cannot distinguish 5-hmC from 5-mC11. To unravel the biology of 5-hmC, we have developed a highly efficient and selective chemical approach to label and capture 5-hmC, taking advantage of a bacteriophage enzyme that adds a glucose moiety to 5-hmC specifically12.
Here we describe a straightforward two-step procedure for selective chemical labeling of 5-hmC. In the first labeling step, 5-hmC in genomic DNA is labeled with a 6-azide-glucose catalyzed by &beta;-GT, a glucosyltransferase from T4 bacteriophage, in a way that transfers the 6-azide-glucose to 5-hmC from the modified cofactor, UDP-6-N3-Glc (6-N3UDPG). In the second step, biotinylation, a disulfide biotin linker is attached to the azide group by click chemistry. Both steps are highly specific and efficient, leading to complete labeling regardless of the abundance of 5-hmC in genomic regions and giving extremely low background. Following biotinylation of 5-hmC, the 5-hmC-containing DNA fragments are then selectively captured using streptavidin beads in a density-independent manner. The resulting 5-hmC-enriched DNA fragments could be used for downstream analyses, including next-generation sequencing.
Our selective labeling and capture protocol confers high sensitivity, applicable to any source of genomic DNA with variable/diverse 5-hmC abundances. Although the main purpose of this protocol is its downstream application (i.e., next-generation sequencing to map out the 5-hmC distribution in genome), it is compatible with single-molecule, real-time SMRT (DNA) sequencing, which is capable of delivering single-base resolution sequencing of 5-hmC.

Described is a two-step labeling process using &beta;-glucosyltransferase (&beta;-GT) to transfer an azide-glucose to 5-hmC, followed by click chemistry to transfer a biotin linker for easy and density-independent enrichment. This efficient and specific labeling method enables enrichment of 5-hmC with extremely low background and high-throughput epigenomic mapping via next-generation sequencing.

Captura selectiva de 5-hidroximetilcitosina a partir de ADN genómico

5-methylcytosine (5-mC) constitutes ~2-8% of the total cytosines in human genomic DNA and impacts a broad range of biological functions, including gene ...

Real-time Imaging of Single Engineered RNA Transcripts in Living Cells Using Ratiometric Bimolecular Beacons

The growing realization that both the temporal and spatial regulation of gene expression can have important consequences on cell function has led to the development of diverse techniques to visualize individual RNA transcripts in single living cells. One promising technique that has recently been described utilizes an oligonucleotide-based optical probe, ratiometric bimolecular beacon (RBMB), to detect RNA transcripts that were engineered to contain at least four tandem repeats of the RBMB target sequence in the 3&#8217;-untranslated region. RBMBs are specifically designed to emit a bright fluorescent signal upon hybridization to complementary RNA, but otherwise remain quenched. The use of a synthetic probe in this approach allows photostable, red-shifted, and highly emissive organic dyes to be used for imaging. Binding of multiple RBMBs to the engineered RNA transcripts results in discrete fluorescence spots when viewed under a wide-field fluorescent microscope. Consequently, the movement of individual RNA transcripts can be readily visualized in real-time by taking a time series of fluorescent images. Here we describe the preparation and purification of RBMBs, delivery into cells by microporation and live-cell imaging of single RNA transcripts.

Ratiometric bimolecular beacons (RBMBs) can be used to image single engineered RNA transcripts in living cells. Here, we describe the preparation and purification of RBMBs, delivery of RBMBs into cells by microporation and fluorescent imaging of single RNA transcripts in real-time.

Imágenes en tiempo real de Soltero Engineered ARN transcritos en células vivas utilizando Ratiometric bimoleculares Balizas

The growing realization that both the temporal and spatial regulation of gene expression can have important consequences on cell function has led to the ...

Fluorescence Based Primer Extension Technique to Determine Transcriptional Starting Points and Cleavage Sites of RNases In Vivo

Fluorescence based primer extension (FPE) is a molecular method to determine transcriptional starting points or processing sites of RNA molecules. This is achieved by reverse transcription of the RNA of interest using specific fluorescently labeled primers and subsequent analysis of the resulting cDNA fragments by denaturing polyacrylamide gel electrophoresis. Simultaneously, a traditional Sanger sequencing reaction is run on the gel to map the ends of the cDNA fragments to their exact corresponding bases. In contrast to 5&#39;-RACE (Rapid Amplification of cDNA Ends), where the product must be cloned and multiple candidates sequenced, the bulk of cDNA fragments generated by primer extension can be simultaneously detected in one gel run. In addition, the whole procedure (from reverse transcription to final analysis of the results) can be completed in one working day. By using fluorescently labeled primers, the use of hazardous radioactive isotope labeled reagents can be avoided and processing times are reduced as products can be detected during the electrophoresis procedure.
In the following protocol, we describe an in vivo fluorescent primer extension method to reliably and rapidly detect the 5&#39; ends of RNAs to deduce transcriptional starting points and RNA processing sites (e.g., by toxin-antitoxin system components) in S. aureus, E. coli and other bacteria.

Fluorescence Based Primer Extension Technique to Determine Transcriptional Starting Points and Cleavage Sites of RNases In Vivo

We here describe a fluorescence based primer extension method to determine transcriptional starting points from bacterial transcripts and RNA processing in vivo using an automated gel sequencer.

Fluorescencia Basado Técnica de extensión del cebador para determinar la transcripción puntos de partida y los sitios de escisión de RNasas<em&gt; En Vivo</em

Fluorescence based primer extension (FPE) is a molecular method to determine transcriptional starting points or processing sites of RNA molecules. This is ...

Targeted DNA Methylation Analysis by Next-generation Sequencing

The role of epigenetic processes in the control of gene expression has been known for a number of years. DNA methylation at cytosine residues is of particular interest for epigenetic studies as it has been demonstrated to be both a long lasting and a dynamic regulator of gene expression. Efforts to examine epigenetic changes in health and disease have been hindered by the lack of high-throughput, quantitatively accurate methods. With the advent and popularization of next-generation sequencing (NGS) technologies, these tools are now being applied to epigenomics in addition to existing genomic and transcriptomic methodologies. For epigenetic investigations of cytosine methylation where regions of interest, such as specific gene promoters or CpG islands, have been identified and there is a need to examine significant numbers of samples with high quantitative accuracy, we have developed a method called Bisulfite Amplicon Sequencing (BSAS). This method combines bisulfite conversion with targeted amplification of regions of interest, transposome-mediated library construction and benchtop NGS. BSAS offers a rapid and efficient method for analysis of up to 10 kb of targeted regions in up to 96 samples at a time that can be performed by most research groups with basic molecular biology skills. The results provide absolute quantitation of cytosine methylation with base specificity. BSAS can be applied to any genomic region from any DNA source. This method is useful for hypothesis testing studies of target regions of interest as well as confirmation of regions identified in genome-wide methylation analyses such as whole genome bisulfite sequencing, reduced representation bisulfite sequencing, and methylated DNA immunoprecipitation sequencing.

Bisulfite amplicon sequencing (BSAS) is a method for quantifying cytosine methylation in targeted genomic regions of interest. This method uses bisulfite conversion paired with PCR amplification of target regions prior to next-generation sequencing to produce absolute quantitation of DNA methylation at a base-specific level.

Dirigida análisis de metilación del ADN mediante secuenciación de próxima generación

The role of epigenetic processes in the control of gene expression has been known for a number of years. DNA methylation at cytosine residues is of ...

RNA Isolation from Cell Specific Subpopulations Using Laser-capture Microdissection Combined with Rapid Immunolabeling

Laser capture microdissection (LCM) allows the isolation of specific cells from thin tissue sections with high spatial resolution. Effective LCM requires precise identification of cells subpopulations from a heterogeneous tissue. Identification of cells of interest for LCM is usually based on morphological criteria or on fluorescent protein reporters. The combination of LCM and rapid immunolabeling offers an alternative and efficient means to visualize specific cell types and to isolate them from surrounding tissue. High-quality RNA can then be extracted from a pure cell population and further processed for downstream applications, including RNA-sequencing, microarray or qRT-PCR. This approach has been previously performed and briefly described in few publications. The goal of this article is to illustrate how to perform rapid immunolabeling of a cell population while keeping RNA integrity, and how to isolate these specific cells using LCM. Herein, we illustrated this multi-step procedure by immunolabeling and capturing dopaminergic cells in brain tissue from one-day-old mice. We highlight key critical steps that deserve special consideration. This protocol can be adapted to a variety of tissues and cells of interest. Researchers from different fields will likely benefit from the demonstration of this approach.

Gene expression analysis of a subset of cells in a specific tissue represents a major challenge. This article describes how to isolate high-quality total RNA from a specific cell population by combining a rapid immunolabeling method with laser capture microdissection.

Aislamiento de ARN a partir de células subpoblaciones específicas utilizando láser captura Microdissection Combinado con Rapid Immunolabeling

Laser capture microdissection (LCM) allows the isolation of specific cells from thin tissue sections with high spatial resolution. Effective LCM requires ...