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.

<table><tbody><tr><td>AmpliTaq Gold polymerase with GeneAmp PCR buffer II and MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Applied Biosystems</td><td>N8080241</td><td></td></tr><tr><td>QIAGEN MinElute PCR purification kit</td><td>Qiagen</td><td>28004</td><td></td></tr><tr><td>M-270 streptavidin beads</td><td>Invitrogen</td><td>653.05</td><td></td></tr><tr><td>Tween-20</td><td>Sigma-Aldrich</td><td>P2287</td><td></td></tr><tr><td>DynaMag-2 magnetic particle separator</td><td>Invitrogen</td><td>120.02D</td><td></td></tr></tbody></table>

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

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

20.1K 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

G2-seq: A High Throughput Sequencing-based Technique for Identifying Late Replicating Regions of the Genome

Numerous techniques have been developed to follow the progress of DNA replication through the S phase of the cell cycle. Most of these techniques have been directed toward elucidation of the location and timing of initiation of genome duplication rather than its completion. However, it is critical that we understand regions of the genome that are last to complete replication, because these regions suffer elevated levels of chromosomal breakage and mutation, and they have been associated with both disease and aging. Here we describe how we have extended a technique that has been used to monitor replication initiation to instead identify those regions of the genome last to complete replication. This approach is based on a combination of flow cytometry and high throughput sequencing. Although this report focuses on the application of this technique to yeast, the approach can be used with any cells that can be sorted in a flow cytometer according to DNA content.

We describe a technique for combining flow cytometry and high throughput sequencing to identify late replicating regions of the genome.

G2-seq: Un'elevato Throughput sequenziamento-base tecnica per identificare replica ritardata regioni del genoma

Numerous techniques have been developed to follow the progress of DNA replication through the S phase of the cell cycle. Most of these techniques have ...

Ricerca

Genetica

Adaptation of Hybridization Capture of Chromatin-associated Proteins for Proteomics to Mammalian Cells

The hybridization capture of chromatin-associated proteins for proteomics (HyCCAPP) technology was initially developed to uncover novel DNA-protein interactions in yeast. It allows analysis of a target region of interest without the need for prior knowledge about likely proteins bound to the target region. This, in theory, allows HyCCAPP to be used to analyze any genomic region of interest, and it provides sufficient flexibility to work in different cell systems. This method is not meant to study binding sites of known transcription factors, a task better suited for Chromatin Immunoprecipitation (ChIP) and ChIP-like methods. The strength of HyCCAPP lies in its ability to explore DNA regions for which there is limited or no knowledge about the proteins bound to it. It can also be a convenient method to avoid biases (present in ChIP-like methods) introduced by protein-based chromatin enrichment using antibodies. Potentially, HyCCAPP can be a powerful tool to uncover truly novel DNA-protein interactions. To date, the technology has been predominantly applied to yeast cells or to high copy repeat sequences in mammalian cells. In order to become the powerful tool we envision, HyCCAPP approaches need to be optimized to efficiently capture single-copy loci in mammalian cells. Here, we present our adaptation of the initial yeast HyCCAPP capture protocol to human cell lines, and show that single-copy chromatin regions can be efficiently isolated with this modified protocol.

This is a method to identify novel DNA-interacting proteins at specific target loci, relying on sequence-specific capture of crosslinked chromatin for subsequent proteomic analyses. No prior knowledge about potential binding proteins, nor cell modifications are required. Initially developed for yeast, the technology has now been adapted for mammalian cells.

Adattamento di ibridazione catturare delle proteine della cromatina associate per proteomica per cellule di mammifero

The hybridization capture of chromatin-associated proteins for proteomics (HyCCAPP) technology was initially developed to uncover novel DNA-protein ...

Promoter Capture Hi-C: High-resolution, Genome-wide Profiling of Promoter Interactions

The three-dimensional organization of the genome is linked to its function. For example, regulatory elements such as transcriptional enhancers control the spatio-temporal expression of their target genes through physical contact, often bridging considerable (in some cases hundreds of kilobases) genomic distances and bypassing nearby genes. The human genome harbors an estimated one million enhancers, the vast majority of which have unknown gene targets. Assigning distal regulatory regions to their target genes is thus crucial to understand gene expression control. We developed Promoter Capture Hi-C (PCHi-C) to enable the genome-wide detection of distal promoter-interacting regions (PIRs), for all promoters in a single experiment. In PCHi-C, highly complex Hi-C libraries are specifically enriched for promoter sequences through in-solution hybrid selection with thousands of biotinylated RNA baits complementary to the ends of all promoter-containing restriction fragments. The aim is to then pull-down promoter sequences and their frequent interaction partners such as enhancers and other potential regulatory elements. After high-throughput paired-end sequencing, a statistical test is applied to each promoter-ligated restriction fragment to identify significant PIRs at the restriction fragment level. We have used PCHi-C to generate an atlas of long-range promoter interactions in dozens of human and mouse cell types. These promoter interactome maps have contributed to a greater understanding of mammalian gene expression control by assigning putative regulatory regions to their target genes and revealing preferential spatial promoter-promoter interaction networks. This information also has high relevance to understanding human genetic disease and the identification of potential disease genes, by linking non-coding disease-associated sequence variants in or near control sequences to their target genes.

DNA regulatory elements, such as enhancers, control gene expression by physically contacting target gene promoters, often through long-range chromosomal interactions spanning large genomic distances. Promoter Capture Hi-C (PCHi-C) identifies significant interactions between promoters and distal regions, enabling the assignment of potential regulatory sequences to their target genes.

Promotore cattura Ciao-c: Ad alta risoluzione, Genome-wide profilatura delle interazioni del promotore

The three-dimensional organization of the genome is linked to its function. For example, regulatory elements such as transcriptional enhancers control the ...

Rare Event Detection Using Error-corrected DNA and RNA Sequencing

Conventional next-generation sequencing techniques (NGS) have allowed for immense genomic characterization for over a decade. Specifically, NGS has been used to analyze the spectrum of clonal mutations in malignancy. Though far more efficient than traditional Sanger methods, NGS struggles with identifying rare clonal and subclonal mutations due to its high error rate of ~0.5&#8211;2.0%. Thus, standard NGS has a limit of detection for mutations that are &#62;0.02 variant allele fraction (VAF). While the clinical significance for mutations this rare in patients without known disease remains unclear, patients treated for leukemia have significantly improved outcomes when residual disease is &#60;0.0001 by flow cytometry. In order to mitigate this artefactual background of NGS, numerous methods have been developed. Here we describe a method for Error-corrected DNA and RNA Sequencing (ECS), which involves tagging individual molecules with both a 16 bp random index for error-correction and an 8 bp patient-specific index for multiplexing. Our method can detect and track clonal mutations at variant allele fractions (VAFs) two orders of magnitude lower than the detection limit of NGS and as rare as 0.0001 VAF.

Next-generation sequencing (NGS) is a powerful tool for genomic characterization that is limited by the high error rate of the platform (~0.5&#8211;2.0%). We describe our methods of error-corrected sequencing that allow us to obviate the NGS error rate and detect mutations at variant allele fractions as rare as 0.0001.

Rilevamento di eventi rari utilizzando la sequenza di RNA e DNA di correzione d'errore

Conventional next-generation sequencing techniques (NGS) have allowed for immense genomic characterization for over a decade. Specifically, NGS has been ...

High-throughput Identification of Gene Regulatory Sequences Using Next-generation Sequencing of Circular Chromosome Conformation Capture (4C-seq)

The identification of regulatory elements for a given target gene poses a significant technical challenge owing to the variability in the positioning and effect sizes of regulatory elements to a target gene. Some progress has been made with the bioinformatic prediction of the existence and function of proximal epigenetic modifications associated with activated gene expression using conserved transcription factor binding sites. Chromatin conformation capture studies have revolutionized our ability to discover physical chromatin contacts between sequences and even within an entire genome. Circular chromatin conformation capture coupled with next-generation sequencing (4C-seq), in particular, is designed to discover all possible physical chromatin interactions for a given sequence of interest (viewpoint), such as a target gene or a regulatory enhancer. Current 4C-seq strategies directly sequence from within the viewpoint but require numerous and diverse viewpoints to be simultaneously sequenced to avoid the technical challenges of uniform base calling (imaging) with next generation sequencing platforms. This volume of experiments may not be practical for many laboratories. Here, we report a modified approach to the 4C-seq protocol that incorporates both an additional restriction enzyme digest and qPCR-based amplification steps that are designed to facilitate a greater capture of diverse sequence reads and mitigate the potential for PCR bias, respectively. Our modified 4C method is amenable to the standard molecular biology lab for assessing chromatin architecture.

High-throughput Identification of Gene Regulatory Sequences Using Next-generation Sequencing of Circular Chromosome Conformation Capture 4C-seq

The identification of physical interactions between genes and regulatory elements is challenging but has been facilitated by chromosome conformation capture methods. This modification to the 4C-seq protocol mitigates PCR bias by minimizing over-amplification of PCR templates and maximizes the mappability of reads by incorporating an addition restriction enzyme digest step.

Identificazione di High-throughput di sequenze regolatorie del Gene mediante sequenziamento di nuova generazione di cromosoma circolare conformazione catturare (4C-seq)

The identification of regulatory elements for a given target gene poses a significant technical challenge owing to the variability in the positioning and ...

Capturing Chromosome Conformation Across Length Scales

Chromosome conformation capture (3C) is used to detect three-dimensional chromatin interactions. Typically, chemical crosslinking with formaldehyde (FA) is used to fix chromatin interactions. Then, chromatin digestion with a restriction enzyme and subsequent religation of fragment ends converts three-dimensional (3D) proximity into unique ligation products. Finally, after reversal of crosslinks, protein removal, and DNA isolation, DNA is sheared and prepared for high-throughput sequencing. The frequency of proximity ligation of pairs of loci is a measure of the frequency of their colocalization in three-dimensional space in a cell population.
A sequenced Hi-C library provides genome-wide information on interaction frequencies between all pairs of loci. The resolution and precision of Hi-C relies on efficient crosslinking that maintains chromatin contacts and frequent and uniform fragmentation of the chromatin. This paper describes an improved in situ Hi-C protocol, Hi-C 3.0, that increases the efficiency of crosslinking by combining two crosslinkers (formaldehyde [FA] and disuccinimidyl glutarate [DSG]), followed by finer digestion using two restriction enzymes (DpnII and DdeI). Hi-C 3.0 is a single protocol for the accurate quantification of genome folding features at smaller scales such as loops and topologically associating domains (TADs), as well as features at larger nucleus-wide scales such as compartments.

Hi-C 3.0 is an improved Hi-C protocol that combines formaldehyde and disuccinimidyl glutarate crosslinkers with a cocktail of DpnII and DdeI restriction enzymes to increase the signal-to-noise ratio and the resolution of chromatin interaction detection.

Acquisizione della conformazione cromosomica su scale di lunghezza

Chromosome conformation capture (3C) is used to detect three-dimensional chromatin interactions. Typically, chemical crosslinking with formaldehyde (FA) ...

<meta charset="utf8"></head><body>Rilevamento di eventi di scissione CRISPR-Cas9 fuori bersaglio con CIRCLE-Seq

CIRCLE-Seq for Interrogation of Off-Target Gene Editing

Circularization for In Vitro Reporting of Cleavage Effects by Sequencing (CIRCLE-seq) is a novel technique developed for the impartial identification of unintended cleavage sites of CRISPR-Cas9 through targeted sequencing of CRISPR-Cas9 cleaved DNA. The protocol involves circularizing genomic DNA (gDNA), which is subsequently treated with the Cas9 protein and a guide RNA (gRNA) of interest. Following treatment, the cleaved DNA is purified and prepared as a library for Illumina sequencing. The sequencing process generates paired-end reads, offering comprehensive data on each cleavage site. CIRCLE-seq provides several advantages over other in vitro methods, including minimal sequencing depth requirements, low background, and high enrichment for Cas9-cleaved gDNA. These advantages enhance sensitivity in identifying both intended and unintended cleavage events. This study provides a comprehensive, step-by-step procedure for examining the off-target activity of CRISPR-Cas9 using CIRCLE-seq. As an example, this protocol is validated by mapping genome-wide unintended cleavage sites of CRISPR-Cas9 during the modification of the AAVS1 locus. The entire CIRCLE-seq process can be completed in two weeks, allowing sufficient time for cell growth, DNA purification, library preparation, and Illumina sequencing. The input of sequencing data into the CIRCLE-seq pipeline facilitates streamlined interpretation and analysis of cleavage sites.

<meta charset="utf8"></head><body>CIRCLE-Seq per l'interrogazione dell'editing genetico off-target

A significant barrier to technologies like CRISPR is the off-target events that can disrupt vital genes. 'Circularization for In Vitro Reporting of Cleavage Effects by Sequencing' (CIRCLE-seq) is a technique designed to identify unintended cleavage sites. This method maps the genome-wide activity of CRISPR-Cas9 with high sensitivity and without bias.

CIRCLE-Seq per l'interrogazione dell'editing genetico off-target

Circularization for In Vitro Reporting of Cleavage Effects by Sequencing (CIRCLE-seq) is a novel technique developed for the impartial identification of ...

gDNA Enrichment by a Transposase-based Technology for NGS Analysis of the Whole Sequence of BRCA1, BRCA2, and 9 Genes Involved in DNA Damage Repair

The widespread use of Next Generation Sequencing has opened up new avenues for cancer research and diagnosis. NGS will bring huge amounts of new data on cancer, and especially cancer genetics. Current knowledge and future discoveries will make it necessary to study a huge number of genes that could be involved in a genetic predisposition to cancer. In this regard, we developed a Nextera design to study 11 complete genes involved in DNA damage repair. This protocol was developed to safely study 11 genes (ATM, BARD1, BRCA1, BRCA2, BRIP1, CHEK2, PALB2, RAD50, RAD51C, RAD80, and TP53) from promoter to 3&#39;-UTR in 24 patients simultaneously. This protocol, based on transposase technology and gDNA enrichment, gives a great advantage in terms of time for the genetic diagnosis thanks to sample multiplexing. This protocol can be safely used with blood gDNA.

gDNA Enrichment by a Transposase-based Technology for NGS Analysis of the Whole Sequence of BRCA1, BRCA2, and 9 Genes Involved in DNA Damage Repair

gDNA enrichment for NGS sequencing is an easy and powerful tool for the study of constitutional mutations. In this article, we present the procedure to analyse simply the complete sequence of 11 genes involved in DNA damage repair.

gDNA arricchimento da una tecnologia basata trasposasi-NGS per l&#39;analisi della sequenza intera<em&gt; BRCA1</em&gt;,<em&gt; BRCA2</em&gt;, e 9 geni coinvolti nella riparazione del DNA Damage

The widespread use of Next Generation Sequencing has opened up new avenues for cancer research and diagnosis. NGS will bring huge amounts of new data on ...

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

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CIRCLE-Seq per l'interrogazione dell'editing genetico off-target

CIRCLE-Seq per l'interrogazione dell'editing genetico off-target

CIRCLE-Seq per l'interrogazione dell'editing genetico off-target

gDNA arricchimento da una tecnologia basata trasposasi-NGS per l'analisi della sequenza intera