Ringraziamo M. Meyer per un consiglio; L&#39;Accademia Croata delle Scienze e delle Arti e il Berlino-Brandeburgo Accademia delle Scienze per il supporto logistico e scientifico, e il Fondo per l&#39;Innovazione del Presidente della Max Planck Society per il sostegno finanziario. JMG è stato sostenuto da una borsa post-dottorato internazionale NSF (OISE-0754461). Sequenze sono depositate presso la banca dati EBI nucleotide con i numeri di adesione: Neandertal 1 (Feldhofer 1) FM865407, Neandertal 2 (Feldhofer 2) FM865408, Sidron 1253 FM865409, Vindija 33,25 FM865410, Mezmaiskaya 1 FM865411

Questo metodo è stato utilizzato nella ricerca riportata in <a href="http://www.sciencemag.org/cgi/content/full/325/5938/318">Briggs et al. Science 325 (5938). 318-321 (2009)</a> . Reagenti richiesti: <ul><li> AmpliTaq Gold DNA polimerasi </li><li> 10x GeneAmp PCR Buffer II </li><li> MgCl2 25mM </li><li> dNTP mix, 25 mm ciascuno </li><li> BSA, 10 mg ml-1 in acqua </li><li> Biologia molecolare acqua di grado </li><li> EB tampone (fornito con il kit MinElute purificazione PCR), 10 mM Tris HCl, pH 8,5 </li><li> MinElute PCR Purification Kit </li><li> M-270 streptavidina Dynabeads </li><li> 2x Binding e Wash (BW) buffer (2 M di NaCl, 10 mM Tris-Cl, 1 mM EDTA, pH 8.0, 0.2% Tween 20) </li><li> 1x Binding e Wash (BW) buffer (2x tampone 2X BindWash diluito in acqua) </li><li> Hot Wash (HW) buffer (2,5 mm MgCl, 1X Taq Oro tampone, 0,1% Tween 20) </li></ul> Per informazioni sul produttore in materia di non-standard reagenti e attrezzature vediamo dopo tavola. 1) Biblioteca di amplificazione <ol><li> Il modello di DNA qui è una libreria di 454 preparata da una fonte a basso numero di copie del DNA, come descritto in (Rohland e Hofreiter, 2007a, 2007b) e (Maricic e Pääbo, 2009). Per amplificare l&#39;intera biblioteca, preparare l&#39;impasto seguenti PCR: <table border="1" cellspacing="0" cellpadding="0"><tbody><tr><td> Reagente </td><td> microlitri per reazione </td></tr><tr><td> Acqua </td><td> 45 </td></tr><tr><td> 10X Gene Amp PCR tampone 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> dNTPs (25 mm ciascuno) </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> AmpliTaq Gold DNA polimerasi (5 U / mL) </td><td> 1 </td></tr><tr><td> 454 DNA stampo biblioteca </td><td> 25 </td></tr><tr><td> Totale </td><td> 100 </td></tr></tbody></table></li><li> Utilizza il seguente programma PCR in un Thermo cycler per 14 cicli di amplificazione <ol><li> 95 ° C 12min </li><li> 95 ° C &#39;30 </li><li> 60 ° C (temperatura di ricottura o desiderato) 1 min </li><li> 72 ° C 1 min </li><li> Vai a 2 (x 13) </li><li> 72 ° C 5min </li><li> 10 ° C ∞ </li></ol></li><li> Purificare la reazione sopra una colonna Qiagen giro MinElute secondo le istruzioni del produttore. Eluire in 50 microlitri di buffer EB. </li><li> Quantificare il prodotto purificato amplificato e un aliquota della biblioteca non amplificata con qPCR (Meyer et al., 2007). Se il prodotto amplificato ha più di 1 X 10 12 copie per ul, ridurre la quantità di modello nella fase successiva estensione Primer per garantire che non più di 2 x 10 12 copie vengono aggiunti alla reazione di primer extension. </li></ol> 2) Primer Extension <ol><li> Preparare una master mix per il numero di reazioni. <table border="0" cellspacing="0" cellpadding="0"><tbody><tr><td> Reagente </td><td> microlitri per reazione </td></tr><tr><td> Acqua </td><td> 45 </td></tr><tr><td> 10X Gene Amp PCR tampone 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> dNTPs (25 mm ciascuno) </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> AmpliTaq Gold DNA polimerasi (5 U / mL) </td><td> 1 </td></tr><tr><td> 454 DNA stampo biblioteca </td><td> 25 </td></tr><tr><td> Totale </td><td> 100 </td></tr></tbody></table></li><li> Eseguire il seguente programma in un termociclatore per la reazione singolo primer extension: <ol><li> 95 ° C 12min </li><li> 60 ° C (o il vostro PEC fondo ricottura temperatura se diverso) 1 min </li><li> 72 ° C 5 min </li><li> 72 ° C per sempre! PUNTO CRITICO Mantenere reazione dopo l&#39;estensione a 72 ° C. Poi direttamente pipetta 150ul PBI o PB tampone per la depurazione QIAGEN MinElute nei tubi prima di togliere dal blocco di calore. Questo è importante per evitare di non-specifico fondo di ricottura e catturare il composto si raffredda. </li></ol></li><li> Purificare la reazione sopra una colonna Qiagen giro MinElute, secondo le istruzioni del produttore. Eluire in 50 microlitri EB. </li></ol> 3) estensione Capture prodotto <ol><li> Risospendere soluzione madre di M-270 perle vortexando. Estrarre 25 microlitri sospensione tallone per campione. Lavare due volte con le perline 500ul 2x BW buffer e risospendere in 25 microlitri di buffer 2xBW per campione. </li><li> Aggiungere 25 microlitri eluato dal passo 2,3-25 microlitri sospensione perle dal punto 3.1. Mescolare poi ruotare per 15 minuti a temperatura ambiente. Raccomandati: tenere restante 5 ul dell&#39;eluato dal punto 2.3 per la quantificazione qPCR. </li><li> Trasferire il composto tutto in una nuova provetta 1,5 ml (questo aiuta a ridurre il riporto di non bersaglio frammenti biblioteca). Pellet il surnatante con il collettore di particelle magnetiche (MPC) e scartare il surnatante. </li><li> Lavare 5 volte con 500 microlitri di buffer 1xBW (molti lavaggi aiutano a rimuovere come sfondo il più possibile). Dopo la fase di lavaggio scorso, centrifugare brevemente e rimuovere le ultime tracce di sopranatante. </li><li> Aggiungere 500μl 1x Hot Wash (HW) buffer. Agitare per 2 minuti a 65 ° C (o 5C sopra il primer PEC Tm) su un blocco termico. Rimuovere il surnatante subito dopo questo, per ridurre al minimo il raffreddamento. (Questo passaggio è quello di rimuovere i frammenti di fondo ancora associato con primer PEC, ma non di fatto esteso durante la fase di estensione). Rimuovere le ultime tracce di sopranatante. </li><li> Risospendere il pellet tallone in 30 microlitri di buffer EB. Trasferire il composto tutto in una nuova provetta 1,5 ml (questo aiuta a ridurre il riporto di non bersaglio frammenti biblioteca). Incubare a 95 ° C per 3 minuti in un termociclatore per l&#39;eluizione. Posto nella MPC e rimuovere il supernatante, avendo cura di lasciare alle spalle le perline. </li></ol> 4) Acquisizione di amplificazione del prodotto <ol><li> Preparare una master mix PCR per il numero di campioni. <table border="1" cellspacing="0" cellpadding="0"><tbody><tr><td> Reagente </td><td> microlitri per reazione </td></tr><tr><td> Acqua </td><td> 45 </td></tr><tr><td> 10X Gene Amp PCR tampone 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> dNTPs (25 mm ciascuno) </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> AmpliTaq Gold DNA polimerasi (5 U / mL) </td><td> 1 </td></tr><tr><td> L&#39;acquisizione dei prodotti eluiti </td><td> 25 </td></tr><tr><td> Totale </td><td> 100 </td></tr></tbody></table></li><li> Utilizza il seguente programma PCR in un Thermo cycler per 14 cicli di amplificazione: <ol><li> 95 ° C 12min </li><li> 95 ° C &#39;30 </li><li> 60 ° C (temperatura di ricottura o desiderato) 1 min </li><li> 72 ° C 1 min </li><li> Vai a 2 (x 13) </li><li> 72 ° C 5min </li><li> 10 ° C ∞ </li></ol></li><li> Purificare la reazione sopra una colonna di silice giro Qiagen MinElute secondo le istruzioni del produttore. Eluire in 50 microlitri di buffer EB. Il prodotto può essere ora utilizzato sia per un secondo round di cattura, a partire dal punto 2.1, o inserito direttamente in emulsione 454 Protocollo PCR, per il sequenziamento. </li></ol> Rappresentante dei risultati: E &#39;fortemente consigliato quando si inizia con il protocollo PEC per effettuare prima una reazione di cattura in cui una piccola quantità di una&#39; sequenza positiva obiettivo di controllo &#39;(ad esempio un ~ 100bp prodotto di PCR - 1 picogrammo è abbastanza facilmente) si mescola con il normale raccomandata quantità di amplificato 454 Template Library (vedi punto 2.1), e la cattura viene eseguita con un primer singolo PEC progettato per catturare il prodotto controllo positivo. Se questa reazione viene eseguito il controllo, qPCR con entrambi positivi di controllo specifico e 454 adattatore specifico per coppie di primer può essere usato per quantificare la quantità di &#39;obiettivo di controllo&#39; contro di fondo nella reazione fondo purificato estensione (1,3), estensione del prodotto Primer (2,3 ) ed eluita prodotto di acquisizione tallone (3,6). In questo modo, sia l&#39;efficacia della rimozione di fondo (&quot;specificità&quot;) e l&#39;efficienza di recupero di destinazione (&quot;sensibilità&quot;) nella vostra condizioni sperimentali possono essere misurati direttamente. Un successo PEC protocollo normalmente ha le seguenti caratteristiche - Sensibilità (misurata dalla quantità di target di controllo mantenuto attraverso il protocollo): Importo totale del target di controllo in eluiti prodotto di acquisizione tallone (3,6) = ~ 1-20% del totale in purificato estensione reazione Primer (2,3). Di fondo (misurata da 454 coppia emPCR primer): Importo totale di sfondo in eluita prodotto di acquisizione tallone (3,6) &lt;0,01% del totale in reazione fondo purificato estensione (2,3). Se c&#39;è meno dell&#39;1% del target rimanenti nel prodotto finale, il passo estensione primer può non hanno avuto successo e devono essere esaminati. Se c&#39;è molto di più dello 0,01% del fondo residuo nei prodotti finali, le fasi di lavaggio potrebbe essere stato erroneamente eseguito e deve essere indagato.

<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.

Il metodo di PEC è semplice, veloce, sensibile e specifico. Quindi abbiamo le applicazioni prevedono più autori al di fuori del DNA antico, quali la cattura di piccoli frammenti di RNA da una libreria di RNA, gli interrogatori di variazione strutturale in un campione aggregato o la cattura di 16S (o di altri loci) diversità da un campione metagenomica. Un punto da ricordare è che la sensibilità di cattura si riduce il numero di PEC primer in un multiplex aumenta la cattura di reazione. PEC non è quindi ideale per la cattura delle regioni catturare molto grandi (ad esempio un megabase o più), ma è molto adatto per la cattura delle regioni obiettivo di piccole dimensioni o addirittura posizioni SNP da molti individui in modo rapido.

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<td>AmpliTaq Gold polymerase with GeneAmp PCR buffer II and MgCl2</td>
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<td>QIAGEN MinElute PCR purification kit</td>
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primer extension capture: targeted sequence retrieval from heavily degraded dna sources

Vi presentiamo un metodo di recupero mirati sequenza di DNA da fonti di DNA che sono fortemente degradati e contaminati con il DNA microbico, come è tipico delle ossa antiche. Il metodo riduce notevolmente la distruzione del campione e le richieste relative al sequenziamento diretto della PCR o approcci di sequenziamento shotgun. Abbiamo usato questo metodo per ricostruire l&#39;intero DNA mitocondriale (mtDNA) genomi di cinque Neanderthal provenienti da tutta la loro distribuzione geografica. La diversità genetica del DNA mitocondriale Neanderthal fine è stata di circa tre volte inferiore a quella del contemporaneo esseri umani moderni. Nonché l&#39;analisi dell&#39;evoluzione delle proteine ​​del mtDNA, questi dati suggeriscono che a lungo termine dimensione della popolazione effettiva di Neanderthal era più piccola di quella degli esseri umani moderni e di grandi scimmie esistenti.

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

Vi presentiamo un metodo di recupero mirato antica sequenza di DNA, che abbiamo usato per ricostruire il genoma mitocondriale completo di cinque individui Neandertal. Confronto di queste sequenze con gli esseri umani oggi suggerisce che i Neanderthal avevano un lungo periodo bassa dimensione effettiva della popolazione.

Primer Capture Estensione: Recupero sequenza mirata da fonti DNA fortemente degradate

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 Views.  Max-Planck Institute for Evolutionary Anthropology, Leipzig. Vi presentiamo un metodo di recupero mirato antica sequenza di DNA, che abbiamo usato per ricostruire il genoma mitocondriale completo di cinque individui Neandertal. Confronto di queste sequenze con gli esseri umani oggi suggerisce che i Neanderthal avevano un lungo periodo bassa dimensione effettiva della popolazione.

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.

Recupero degli ovociti mouse

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

Ricerca

Biologia

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.

Proboscide Estensione di risposta (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 ...

Isolation of Genomic DNA from Mouse Tails

Isolamento del DNA genomico da Tails mouse

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-Free Selezione aptamero utilizzando una libreria di DNA casuale

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.

Genomica risonanza magnetica - una risorsa pubblica per lo Studio Patterns sequenza all&#39;interno del DNA genomico

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.

Cattura selettiva di 5 hydroxymethylcytosine dal DNA genomico

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.

In tempo reale Imaging del singolo Engineered RNA trascritti in cellule viventi Utilizzando Ratiometric bimolecolari 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.

Fluorescenza base tecnica Primer Extension determinare trascrizionali punti di partenza e siti di taglio di RNasi<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.

Targeted DNA metilazione Analisi per sequenziamento di prossima generazione

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.

RNA isolamento da cellulari sottopopolazioni specifiche utilizzando laser-capture Microdissection combinazione con Rapid immunomarcatura

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