Nous tenons à remercier M. Meyer pour des conseils; l&#39;Académie croate des Sciences et des Arts et l&#39;Académie de Berlin-Brandebourg des sciences pour le soutien logistique et scientifique, et le Fonds d&#39;innovation de la présidentielle la Société Max Planck pour le soutien financier. JMG a été soutenue par une bourse de recherche postdoctorale de NSF International (OISE-0754461). Les séquences sont déposés à la base de données EBI nucléotides avec des numéros d&#39;adhésion: Neandertal 1 (Feldhofer 1) FM865407, Neandertal 2 (Feldhofer 2) FM865408, Sidron 1253 FM865409, Vindija 33,25 FM865410, Mezmaiskaya une FM865411

Cette méthode a été utilisée dans la recherche présentée dans <a href="http://www.sciencemag.org/cgi/content/full/325/5938/318">Briggs et al., Science 325 (5938). 318-321 (2009)</a> . Réactifs nécessaires: <ul><li> Polymérase AmpliTaq ADN d&#39;or </li><li> 10x GeneAmp PCR Buffer II </li><li> MgCl 2 25 mM </li><li> mélange de dNTP, 25 mM de chaque </li><li> BSA, 10 mg ml-1 dans l&#39;eau </li><li> Biologie moléculaire de qualité de l&#39;eau </li><li> EB tampon (fourni avec le kit MinElute PCR Purification), 10 mM Tris HCl, pH 8,5 </li><li> Kit MinElute PCR Purification </li><li> M-270 streptavidine Dynabeads </li><li> 2x reliure et de lavage (BW) tampon (2 M de NaCl, 10 mM Tris-Cl, EDTA 1 mM, pH 8,0, 0,2% Tween 20) </li><li> 1x reliure et de lavage (BW) tampon (2x BindWash tampon dilué dans de l&#39;eau 2X) </li><li> Laver à chaud (HW) tampon (2,5 mM MgCl, 1X Taq Or tampon, 0,1% Tween 20) </li></ul> Pour plus d&#39;informations concernant la non-fabricant de réactifs et du matériel standard de voir plus loin tableau. Bibliothèque d&#39;amplification 1) <ol><li> La matrice d&#39;ADN est ici une bibliothèque 454 prêts d&#39;une source de copies d&#39;ADN faible nombre tel que décrit dans (Rohland et Hofreiter, 2007a, 2007b) et (Maricic et Pääbo, 2009). Pour amplifier la bibliothèque entière, préparer le mélange de PCR suivant: <table border="1" cellspacing="0" cellpadding="0"><tbody><tr><td> Réactifs </td><td> ul par la réaction </td></tr><tr><td> Eau </td><td> 45 </td></tr><tr><td> Gene Amp PCR 10X tampon II </td><td> 10 </td></tr><tr><td> MgCl 2 (25mm) </td><td> 10 </td></tr><tr><td> BSA (10 mg / ml) </td><td> 2 </td></tr><tr><td> dNTP (25 mM chacun) </td><td> 1 </td></tr><tr><td> 454 emPCR fwd primer/10uM </td><td> 3 </td></tr><tr><td> 454 emPCR rvs primer/10uM </td><td> 3 </td></tr><tr><td> L&#39;ADN polymérase AmpliTaq Gold (5 U / pl) </td><td> 1 </td></tr><tr><td> 454 modèle de banque d&#39;ADN </td><td> 25 </td></tr><tr><td> Total des </td><td> 100 </td></tr></tbody></table></li><li> Utilisez le programme suivant PCR dans un cycleur thermique pour 14 d&#39;amplification des cycles <ol><li> 95 ° C 12min </li><li> 95 ° C 30s </li><li> 60 ° C (température de recuit ou désiré) 1 min </li><li> 72 ° C 1 min </li><li> Aller à 2 (x 13) </li><li> 72 ° C 5min </li><li> 10 ° C ∞ </li></ol></li><li> Purifier la réaction sur une colonne Qiagen MinElute conformément aux instructions du fabricant. Eluer dans 50 pi de tampon EB. </li><li> Quantifier le produit purifié amplifiée ainsi que d&#39;une partie aliquote de la bibliothèque non amplifié par qPCR (Meyer et al., 2007). Si le produit amplifié a plus de 1 X 10 12 copies par UL, réduire la quantité de modèle dans l&#39;étape d&#39;extension d&#39;amorces suivantes pour s&#39;assurer que pas plus de 2 X 10 12 copies sont ajoutés à la réaction extension d&#39;amorce. </li></ol> Extension d&#39;amorce 2) <ol><li> Préparer un master mix pour le nombre requis de réactions. <table border="0" cellspacing="0" cellpadding="0"><tbody><tr><td> Réactifs </td><td> ul par la réaction </td></tr><tr><td> Eau </td><td> 45 </td></tr><tr><td> Gene Amp PCR 10X tampon II </td><td> 10 </td></tr><tr><td> MgCl 2 (25mm) </td><td> 10 </td></tr><tr><td> BSA (10 mg / ml) </td><td> 2 </td></tr><tr><td> dNTP (25 mM chacun) </td><td> 1 </td></tr><tr><td> 454 emPCR fwd primer/10uM </td><td> 3 </td></tr><tr><td> 454 emPCR rvs primer/10uM </td><td> 3 </td></tr><tr><td> L&#39;ADN polymérase AmpliTaq Gold (5 U / pl) </td><td> 1 </td></tr><tr><td> 454 modèle de banque d&#39;ADN </td><td> 25 </td></tr><tr><td> Total des </td><td> 100 </td></tr></tbody></table></li><li> Exécutez le programme suivant dans un thermocycleur pour la réaction d&#39;extension d&#39;amorces unique: <ol><li> 95 ° C 12min </li><li> 60 ° C (ou votre apprêt PEC recuit température si différente) 1 min </li><li> 72 ° C 5 min </li><li> 72 ° C pour toujours! ÉTAPE CRITIQUE Gardez réaction après l&#39;extension à 72 ° C. Puis directement pipette 150ul PBI ou PB tampon pour QIAGEN MinElute de purification dans les tubes avant de retirer le bloc thermique. Ceci est important pour éviter la non-amorce spécifique de recuit et de capturer que le mélange refroidit. </li></ol></li><li> Purifier la réaction sur une colonne Qiagen MinElute, conformément aux instructions du fabricant. Eluer dans 50 EB ul. </li></ol> 3) la capture de produits d&#39;extension <ol><li> Solution stock Resuspendre de M-270 billes par vortex. Sortez 25 ul de suspension de billes par échantillon. Laver les billes à deux reprises avec 500ul de tampon 2x BW et remettre en suspension dans 25 ul de tampon 2xBW par échantillon. </li><li> Ajouter 25 ul éluat de l&#39;étape de 2,3 à 25 pi de suspension de billes étape 3.1. Mélangez puis faites pivoter pour 15 min à température ambiante. Recommandés: conserver restante de 5 ul de l&#39;éluat de l&#39;étape 2.3 pour la quantification par qPCR. </li><li> Transférer le mélange dans son ensemble à un nouveau tube de 1.5ml (cela permet de réduire l&#39;entraînement des fragments de bibliothèque non-cible). Pellet le surnageant en utilisant le collecteur de particules magnétiques (MPC) et jeter le surnageant. </li><li> Laver 5 fois avec 500 ul de tampon 1xBW (nombreux lavages aider à enlever le fond autant que possible). Après la dernière étape de lavage, centrifuger brièvement et éliminer les dernières traces de surnageant. </li><li> Ajouter 500μl 1x chaud Laver (HW) tampon. Agiter pendant 2 min à 65 ° C (ou 5C-dessus de la Tm PEC amorce) sur un bloc thermique. Retirer le surnageant rapidement après cela, afin de minimiser le refroidissement. (Cette étape est d&#39;enlever des fragments de fond reste associé à des amorces PEC, mais pas vraiment étendu sur le cours de l&#39;étape d&#39;extension). Enlevez les dernières traces de surnageant. </li><li> Reprendre le culot de billes dans 30 ul de tampon EB. Transférer le mélange dans son ensemble à un nouveau tube de 1.5ml (cela permet de réduire l&#39;entraînement des fragments de bibliothèque non-cible). Incuber à 95 ° C pendant 3 minutes dans un cycleur thermique pour l&#39;élution. Placer dans les PPM et retirer le surnageant, en prenant soin de laisser les billes. </li></ol> 4) Capture d&#39;amplification produit <ol><li> Préparer un master mix PCR pour le nombre requis d&#39;échantillons. <table border="1" cellspacing="0" cellpadding="0"><tbody><tr><td> Réactifs </td><td> ul par la réaction </td></tr><tr><td> Eau </td><td> 45 </td></tr><tr><td> Gene Amp PCR 10X tampon II </td><td> 10 </td></tr><tr><td> MgCl 2 (25mm) </td><td> 10 </td></tr><tr><td> BSA (10 mg / ml) </td><td> 2 </td></tr><tr><td> dNTP (25 mM chacun) </td><td> 1 </td></tr><tr><td> 454 emPCR fwd primer/10uM </td><td> 3 </td></tr><tr><td> 454 emPCR rvs primer/10uM </td><td> 3 </td></tr><tr><td> L&#39;ADN polymérase AmpliTaq Gold (5 U / pl) </td><td> 1 </td></tr><tr><td> Produit de capture élué </td><td> 25 </td></tr><tr><td> Total des </td><td> 100 </td></tr></tbody></table></li><li> Utilisez le programme suivant PCR dans un cycleur thermique pour 14 d&#39;amplification des cycles: <ol><li> 95 ° C 12min </li><li> 95 ° C 30s </li><li> 60 ° C (température de recuit ou désiré) 1 min </li><li> 72 ° C 1 min </li><li> Aller à 2 (x 13) </li><li> 72 ° C 5min </li><li> 10 ° C ∞ </li></ol></li><li> Purifier la réaction sur une colonne Qiagen MinElute tour de silice selon les instructions du fabricant. Eluer dans 50 ul de tampon EB. Le produit peut être désormais utilisé soit pour un second tour de la capture, à partir de l&#39;étape 2.1, ou entrés directement dans le protocole d&#39;émulsion 454 PCR, pour le séquençage. </li></ol> Les résultats représentatifs: Il est fortement recommandé lors du démarrage avec le protocole PEC à effectuer d&#39;abord une réaction de capture où une petite quantité d&#39;une «séquence cible de maîtrise de positif» (par exemple un ~ 100pb produit PCR - 1 picogramme est assez facilement) est mélangée avec la normale recommandée quantité de 454 amplifié modèle de bibliothèque (voir l&#39;étape 2.1), et la capture est effectuée avec une seule amorce PEC conçu pour capturer le produit de contrôle positif. Si cette réaction de contrôle est effectué, à la fois avec qPCR contrôle positif spécifiques et 454 paires adaptateur amorce spécifique peut être utilisé pour quantifier le montant des «cibles de contrôle» par rapport au fond dans la réaction d&#39;extension d&#39;amorces purifiées (1,3), le produit d&#39;extension d&#39;amorces (2,3 ) et on élue produit de la capture billes (3,6). De cette façon, à la fois l&#39;efficacité de la suppression du fond (la «spécificité») et l&#39;efficacité de la récupération cible («sensibilité») dans votre conditions expérimentales peuvent être mesurées directement. Un succès PEC protocole a normalement les propriétés suivantes - Sensibilité (mesurée par le montant de la cible de maîtrise retenus par le protocole): Montant total de la cible de contrôle dans la capture des produits élués billes (3,6) = ~ 1-20% du montant total de la réaction d&#39;extension d&#39;amorces purifiées (2,3). Contexte (mesurée par 454 paire d&#39;amorces emPCR): Montant total de l&#39;expérience dans la capture des produits élués billes (3,6) &lt;0,01% du montant total en réaction amorce d&#39;extension purifiée (2,3). S&#39;il ya moins de 1% de l&#39;objectif restant dans le produit final, l&#39;étape d&#39;extension d&#39;amorces peuvent avoir été infructueuse et devrait être étudiée. Si il ya beaucoup plus de 0,01% de l&#39;arrière-plan restant dans le produit final, les étapes de lavage peuvent avoir été incorrectement effectuée et devrait être étudiée.

<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.

La méthode PEC est simple, rapide, sensible et spécifique. C&#39;est pourquoi nous les applications multiples auteurs envisagent en dehors d&#39;ADN ancien, comme la capture des petits fragments d&#39;ARN à partir d&#39;une bibliothèque de l&#39;ARN, l&#39;interrogatoire de variation structurelle d&#39;un échantillon groupé ou la capture de 16S (ou d&#39;autres loci) la diversité d&#39;un échantillon de métagénomique. Un point à mentionner est que la sensibilité de capture diminue en fonction du nombre de PEC des amorces dans une réaction augmente multiplexe capture. PEC n&#39;est donc pas idéal pour la capture des régions capture de très grande taille (par exemple, une mégabase ou plus), mais est extrêmement bien adapté pour la capture des régions cibles petites ou même les positions du SNP de nombreuses personnes d&#39;une manière rapide.

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<td>AmpliTaq Gold polymerase with GeneAmp PCR buffer II and MgCl2</td>
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<td>N8080241</td>
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<td>QIAGEN MinElute PCR purification kit</td>
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<td>Qiagen</td>
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<td>M-270 streptavidin beads</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>653.05</td>
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<td>Tween-20</td>
<td>&nbsp;</td>
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<td>P2287</td>
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<td>DynaMag-2 magnetic particle separator</td>
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primer extension capture: targeted sequence retrieval from heavily degraded dna sources

Nous présentons une méthode de récupération séquence d&#39;ADN cible à partir de sources d&#39;ADN qui sont fortement dégradées et contaminées avec de l&#39;ADN microbien, ce qui est typique des ossements anciens. La méthode réduit considérablement la destruction de l&#39;échantillon et les exigences relatives à séquençage PCR directe ou approches de séquençage shotgun. Nous avons utilisé cette méthode pour reconstruire la complète ADN mitochondrial (ADNmt) des génomes de cinq Néandertaliens de partout leur aire géographique. L&#39;ADNmt de la diversité génétique des Néandertaliens du retard a été environ trois fois inférieur à celui des hommes modernes contemporains. Ensemble, avec les analyses de l&#39;évolution des protéines ADNmt, ces données suggèrent que la taille de longue durée effective de la population des Néandertaliens était plus petit que celui des humains modernes et existantes des grands singes.

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

Nous présentons une méthode d&#39;extraction ciblée de séquences d&#39;ADN ancien, qui nous permettent de reconstituer le génome mitochondrial complet de cinq individus néandertaliens. La comparaison de ces séquences avec les humains d&#39;aujourd&#39;hui suggère que les Néandertaliens avaient une longue durée à faible taille de la population effective.

Capturez extension d&#39;amorce: Retrieval séquence ciblée à partir de sources d&#39;ADN fortement dégradées

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 vues.  Max-Planck Institute for Evolutionary Anthropology, Leipzig. Nous présentons une méthode d'extraction ciblée de séquences d'ADN ancien, qui nous permettent de reconstituer le génome mitochondrial complet de cinq individus néandertaliens. La comparaison de ces séquences avec les humains d'aujourd'hui suggère que les Néandertaliens avaient une longue durée à faible taille de la population effective.

Vidéo: 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.

Récupération des ovocytes de souris

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

Recherche

Biologie

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.

Extension Proboscis Réponse (PER) Dosage de la drosophile

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

Isolement de l&#39;ADN génomique de souris Tails

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.

Primaire-Libre Choix aptamères en utilisant une bibliothèque d&#39;ADN aléatoires

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.

Genomic IRM - une ressource publique pour l&#39;étude des schémas de séquence dans l&#39;ADN génomique

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.

Capture d&#39;sélectif de 5-hydroxyméthylcytosine partir de l&#39;ADN génomique

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.

Imagerie en temps réel de l&#39;unité d&#39;ingénierie des ARN transcrits dans des cellules vivantes utilisant Ratiométrique bimoléculaires 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 ...

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.

Fluorescence Basé Technique d&#39;extension d&#39;amorce pour déterminer transcription Points de départ et des sites de clivage de RNases<em&gt; In 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.

Ciblé méthylation de l&#39;ADN Analyse par séquençage de nouvelle génération

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.

Isolement de l&#39;ARN à partir de sous-populations cellulaires spécifiques en utilisant le laser capture microdissection Combiné avec Rapid immunomarquage

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