Questo studio è stato sostenuto in parte dal National Institutes of Health (GM071440 a CH e NS051630/MH076090/MH078972 a PJ).

1. Frammentazione del DNA genomico Frammento di DNA genomico mediante ultrasuoni ad una vasta dimensione desiderata adatto per il genoma a livello di piattaforma di sequenziamento. (Di solito sonicazione a ~ 300 bp.) Verificare la distribuzione dimensionale del DNA genomico frammentato 1% gel (Figura 1). 2. Estrazione del DNA Determinare le quantità di DNA di partenza in base alla abbondanza di 5-HMC nel DNA genomico. Dal 5-HMC livelli variano in modo significativo in diversi tipi di tessuto, quantità di DNA di partenza dipendono dalle 5-HMC livelli dei campioni. Si prega di fare riferimento alla Tabella 1 per gli esempi. 3. β-GT reazione catalizzata (reazione di trasferimento del glucosio) <ol><li> Mescolare pipettando la miscela come descritto in Tabella 2 e incubare a 37 ° C in bagno di acqua per 1 ora. </li><li> Dopo l&#39;incubazione, pulire la reazione con Kit Rimozione QIAquick Nucleotide, con 10 microgrammi di DNA percolonna. Eluire con 30 microlitri di acqua per colonna e combinare. </li></ol> 4. Biotinilazione Reazione (Click Chemistry) <ol><li> Aggiungere DBCO-SS-PEG3-Biotin coniugato soluzione di lavoro (1 mM) nella soluzione di DNA eluito (punto 3) ad una concentrazione finale di 150 pM (cioè 5 ml di soluzione di lavoro per 30 microlitri soluzione di DNA). </li><li> Mescolare pipettando e incubare a 37 ° C bagno di acqua per 2 ore. </li><li> Pulire la reazione con Kit Rimozione QIAquick Nucleotide. Il volume ideale di eluizione totale è di 100 ul. </li><li> Quantificare la quantità di DNA recuperato mediante spettrofotometro scala microlitro (ad es., NanoDrop). </li></ol> 5. Cattura di 5-HMC contenente DNA <ol><li> Lavare 50 pl di Dynabeads MyOne streptavidina C1 3 volte con 1 ml di 1X B &amp; W tampone secondo le istruzioni del produttore. Separare le perline con un supporto magnetico. </li><li> Aggiungere il volume uguale di 2X B &amp; W per il buffer di recupero D biotinilatoNA (100 ul) ai talloni lavate. </li><li> Incubare per 15 minuti a temperatura ambiente con lieve rotazione su un rotatore. </li><li> Separare le perline con un supporto magnetico e lavare le microsfere 3 volte con 1 ml di tampone 1X B &amp; W. </li><li> Eluire il DNA incubando le perle in 100 pl di preparati DTT 50 mM per 2 ore a temperatura ambiente con lieve rotazione su un rotatore. </li><li> Separare le perline con un supporto magnetico. Aspirare il eluente e carico su un Micro Bio-Spin 6 Colonna secondo le istruzioni fabbricazione per rimuovere il DTT. Il DNA bersaglio è nella soluzione ora. </li><li> Purificare il DNA eluito dal passaggio precedente Purification Kit Qiagen MinElute PCR e DNA eluire in 10 microlitri di tampone EB. Quantificare DNA utilizzando fluorimetro Qubit, NanoDrop o se la concentrazione è superiore a 20 ng / ul. Il DNA è pronto per la valle genome-wide preparazione sequenziamento biblioteca. </li></ol> 6. Risultati rappresentativi Se la qualità of DNA genomico è alta, i rendimenti tipici di recupero dopo il β-GT e le reazioni sono biotinilazione ~ 60-70%. Tuttavia, l&#39;efficienza di cattura variare significativamente diversi tipi di tessuto a seconda delle 5-HMC livelli dei campioni. Tipicamente, l&#39;efficienza di cattura per il DNA genomico è cervello ~ 4-9%, e in alcuni casi estremi, l&#39;efficienza può arrivare fino al 12%. Per le cellule ES, l&#39;efficienza di cattura medio è ~ 2-4%, in contrasto con ~ 0,5% per le cellule staminali neuronali. Il più basso rendimento visto finora è stato per il DNA genomico da cellule tumorali. Tutto DNA arricchito è pronto per i protocolli della libreria standard di nuova generazione di preparazione. Inoltre, il DNA catturato può anche essere usato come stampo per PCR in tempo reale per rilevare l&#39;arricchimento di alcuni frammenti rispetto al DNA di ingresso, se i primers correlati sono disponibili. <img alt="Figura 1" src="/files/ftp_upload/4441/4441fig1.jpg" /> Figura 1. Sonicati umani frammenti di DNA genomico in1% gel. 10 ug di DNA genomico isolato da cellule iPS umane in 120 pl di tampone TE 1X stata sonicata utilizzando un dispositivo di sonicazione (Covaris). Dopo sonicazione, 2 microlitri del DNA sonicato viene caricata su gel di agarosio 1% usando 100 bp di DNA marcatore per confrontare le dimensioni dei frammenti di DNA sonicato. <table border="1"><tbody><tr><td> Componente </td><td> Volume </td><td> Concentrazione finale </td></tr><tr><td> Acqua </td><td> _ Pl </td><td></td></tr><tr><td> 10 X β-GT Reaction Buffer </td><td> 2 microlitri </td><td> 1 X </td></tr><tr><td> Fino a 10 microgrammi di DNA genomico </td><td> _ Pl </td><td> Fino a 500 ng / pl </td></tr><tr><td> UDP-6-N 3-Glc (3 mM) </td><td> 0,67 pl </td><td> 100 pM </td></tr><tr><td> β-GT (40 pM) </td><td> 1 ml </td><td> 2 pM </td></tr><tr><td> Volume totale </td><td> 20 pl </td&gt; <td></td></tr></tbody></table> i) Per il DNA genomico del tessuto (alta 5-HMC contenuto&gt; 0,1%) <table border="1"><tbody><tr><td> Componente </td><td> Volume </td><td> Concentrazione finale </td></tr><tr><td> Acqua </td><td> _ Pl </td><td></td></tr><tr><td> 10 X β-GT Reaction Buffer </td><td> 10 pl </td><td> 1 X </td></tr><tr><td> Fino a 20 microgrammi di DNA genomico </td><td> _ Pl </td><td> Fino a 500 ng / pl </td></tr><tr><td> UDP-6-N3-Glc (3 mM) </td><td> 1,33 pl </td><td> 100 pM </td></tr><tr><td> β-GT (40 pM) </td><td> 2 microlitri </td><td> 2 pM </td></tr><tr><td> Volume totale </td><td> 40 ml </td><td></td></tr></tbody></table> ii) Per il DNA genomico di cellule staminali (mediana 5 HMC contenuti ~ 0,05%) <table border="1"><tbody><tr><td> Componente </td><td> Volume </td><td> Concentrazione finale </td></tr><tr><td> Acqua </td><td> _ Pl </td><td></td></tr><tr><td> 10 X β-GT Reaction Buffer </td><td> 10 pl </td><td> 1 X </td></tr><tr><td> Fino a 50 microgrammi di DNA genomico </td><td> _ Pl </td><td> Fino a 500 ng / pl </td></tr><tr><td> UDP-6-N3-Glc (3 mM) </td><td> 3,33 pl </td><td> 100 pM </td></tr><tr><td> β-GT (40 pM) </td><td> 5 microlitri </td><td> 2 pM </td></tr><tr><td> Volume totale </td><td> 100 pl </td><td></td></tr></tbody></table> iii) Per il DNA genomico delle cellule del cancro (basso 5-HMC contenuti ~ 0,01%) Tabella 1. Esempi di quantità di DNA di ingresso e di reazioni etichettatura utilizzando i campioni con diversi livelli di 5-HMC secondo il metodo chimico selettivo etichettatura. <table border="1"><tbody><tr><td> Campione </td><td> 5-HMC livello </strong&gt; </td><td> DNA di partenza (mg) </td><td> Il recupero dopo l&#39;etichettatura (ingresso a sfere) (mg) </td><td> Recupero resa </td><td> Pull-down DNA (ng) </td><td> Pull-down cedere </td></tr><tr><td> Del mouse cervelletto adulto </td><td> 0,4% </td><td> 10 </td><td> 7,5 </td><td> 75% </td><td> 236 </td><td> 3,1% </td></tr><tr><td> Postnatale giorno 7 del mouse cervelletto </td><td> 0,1% </td><td> 11 </td><td> 9 </td><td> 82% </td><td> 140 </td><td> 1,6% </td></tr><tr><td> Topo ES cellule E14 </td><td> 0,05% </td><td> 60 </td><td> 42 </td><td> 70% </td><td> 350 </td><td> 0,8% </td></tr></tbody></table> Tabella 2. Risultati rappresentativi di tessuti cerebrali di topo e cellule ES.

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Nessun conflitto di interessi dichiarati.

5-hydroxymethylcytosine (5-HMC) è un regalo recentemente identificato modificazione epigenetica in quantità notevoli in alcuni tipi di cellule di mammifero. Il metodo qui presentato è per la determinazione del genoma distribuzione a livello di 5-HMC. Usiamo batteriofago T4 β-glucosil trasferire una porzione multistrato glucosio contenente un gruppo azide sul gruppo ossidrile di 5-HMC. Il gruppo azide può essere chimicamente modificato con biotina per il rilevamento, l&#39;arricchimento di affinità, e sequenziament...

<table border="1"><tbody><tr><td> Nome </td><td> Azienda </td><td> Catalogare # </td><td> Commento </td></tr><tr><td></td><td></td><td></td><td> Reagenti </td></tr><tr><td> Cloruro di sodio 5M (NaCl) </td><td> Promega </td><td> V4221 </td><td></td></tr><tr><td> 0.5M pH8.0 Etilendiammintetraacetato (EDTA) </td><td> Promega </td><td> V4231 </td><td></td></tr><tr><td> 1M Trizma base (Tris) pH7.5 </td><td> Invitrogen </td><td> 15567-027) </td><td></td></tr><tr><td> HEPES 1M, pH 7,4 </td><td> Invitrogen </td><td> 15630 </td><td></td></tr><tr><td> Cloruro di magnesio (MgCl2) 1M </td><td> Ambion </td><td> AM9530G </td><td></td></tr><tr><td> Dimetilsolfossido (DMSO) </td><td> Sigma </td><td> D8418 </td><td></td></tr><tr><td> Tween 20 </td><td> Fisher BioReagents </td><td> BP337-100 </td><td></td></tr><tr><td> DBCO-SS-PEG3-biotina coniugato </td><td> Fare clic su Strumenti Chimica </td><td> A112P3 </td><td></td></tr><tr><td> 1,4-Dithiothreitol, ultrapura (DTT) superpuro </td><td> Invitrogen </td><td> 15508-013 </td><td></td></tr><tr><td> QIAquick Kit Rimozione Nucleotide </td><td> Qiagen </td><td> 28304 </td><td></td></tr><tr><td> Micro Bio-Spin 6 Colonna </td><td> Bio-Rad </td><td> 732-6222 </td><td></td></tr><tr><td> Dynabeads MyOne </td><td> Invitrogen </td><td> 650-01 </td><td></td></tr><tr><td> Streptavidina C1 </td><td></td><td></td><td></td></tr><tr><td> Qiagen MinElute PCR Purification Kit </td><td> Qiagen </td><td> 28004 </td><td></td></tr><tr><td> Ultrapura agarosio </td><td> Invitrogen </td><td> 16500500 </td><td></td></tr><tr><td> UDP-6-N 3-glucosio </td><td> Motif attivo </td><td> 55013 </td><td></td></tr><tr><td></td><td></td><td></td><td> Enzima </td></tr><tr><td> β-glucosil (β-GT) </td><td> New England Biolab </td><td> M0357 </td><td></td></tr><tr><td></td><td></td><td></td><td> Attrezzatura </td></tr><tr><td> Sonicazione dispositivo </td><td> Covaris </td><td></td><td></td></tr><tr><td> Desktop centrifuga </td><td></td><td></td><td></td></tr><tr><td> Bagnomaria </td><td> Fisher Scientific </td><td></td><td></td></tr><tr><td> Gel apparecchi in esecuzione </td><td> Bio-Rad </td><td></td><td></td></tr><tr><td> NanoDrop1000 </td><td> Thermo Scientific </td><td></td><td></td></tr><tr><td> Labquake Tubo Shaker </td><td> Barnstead </td><td></td><td></td></tr><tr><td> Labquake Tubo Shaker </td><td> Thermolyne </td><td></td><td></td></tr><tr><td> Separazione supporto magnetico </td><td> Promega </td><td> Z5342 </td><td></td></tr><tr><td> Qubit 2,0 fluorimetro </td><td> Invitrogen </td><td></td&gt; <td></td></tr><tr><td></td><td></td><td></td><td> Reagente di installazione 10 X β-GT Reaction Buffer (500 mM HEPES pH 7,9, 250 mM MgCl2) 2 X Legatura e lavaggio (B &amp; W) buffer (10 mM Tris pH 7.5, 1 mM EDTA, 2 M NaCl, 0,02% Tween 20) . </td></tr></tbody></table>

selective capture of 5-hydroxymethylcytosine from genomic dna

5-metilcitosina (5-mC) costituisce ~ 2-8% dei cytosines totali in DNA genomico umano e influisce un&#39;ampia gamma di funzioni biologiche, tra l&#39;espressione genica, il mantenimento dell&#39;integrità genomica, imprinting parentale, inattivazione del cromosoma X, regolazione sviluppo, invecchiamento e cancro 1. Recentemente, la presenza di un ossidato 5-mC, 5-hydroxymethylcytosine (5-HMC), è stato scoperto in cellule di mammifero, in particolare nel staminali embrionali (ES) cellule e cellule neuronali 2-4. 5-HMC è generato per ossidazione di 5-mC catalizzata da TET famiglia ferro (II) / α-chetoglutarato-dipendente diossigenasi 2, 3. 5-HMC si propone di essere coinvolto nel mantenimento di staminali embrionali (MES) delle cellule, emopoiesi normale e le neoplasie, e zigote sviluppo 2, 5-10. Per capire meglio la funzione di 5-HMC, un sistema di sequenziamento affidabile e semplice è essenziale. Sequenziamento bisolfito tradizionale non è possibile distinguere 5-HMC da 5-MC 11 </sup&gt;. Per svelare la biologia di 5-HMC, abbiamo sviluppato un approccio chimico altamente efficiente e selettivo di etichettare e catturare 5-HMC, approfittando di un enzima batteriofago che aggiunge una frazione di glucosio al 5-HMC specifico 12. Qui si descrive una semplice procedura in due fasi per l&#39;etichettatura chimica selettiva di 5-HMC. Nella prima fase di etichettatura, 5-HMC nel DNA genomico viene etichettato con un 6-catalizzata azide-glucosio da β-GT, un glucosiltransferasi dal batteriofago T4, in modo che trasferisce il 6-azide-glucosio al 5-HMC dal modificato cofattore, UDP-6-N3-Glc (6-N3UDPG). Nella seconda fase, biotinilazione, un linker biotina disolfuro è attaccato al gruppo azide dalla chimica click. Entrambe le fasi sono altamente specifici ed efficienti, portando a completare etichettatura indipendentemente l&#39;abbondanza di 5-HMC in regioni genomiche e dando fondo estremamente bassa. Biotinilazione seguito di 5-HMC, i 5-HMC contenenti frammenti di DNA vengono selettivamente catturatiutilizzando perline streptavidina in una densità modo indipendente. La risultante 5-HMC arricchiti frammenti di DNA potrebbe essere utilizzato per analisi a valle, sequenziamento di prossima generazione. La nostra etichettatura selettiva e il protocollo di acquisizione conferisce elevata sensibilità, applicabile a qualsiasi fonte di DNA genomico con variabili / 5-HMC diverse abbondanze. Anche se lo scopo principale di questo protocollo è la sua applicazione a valle (ad es., Sequenziamento di prossima generazione per tracciare il 5-HMC distribuzione nel genoma), è compatibile con singola molecola, in tempo reale SMRT (DNA) sequenziamento, che è in grado di erogare singola base sequenziamento risoluzione del 5-HMC.

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Selective Capture of 5-hydroxymethylcytosine from Genomic DNA

Descritto è un processo a due fasi usando etichettatura β-glucosil (β-GT) per trasferire una azide-glucosio al 5-HMC, seguita dalla chimica click per trasferire un linker biotina per l&#39;arricchimento facile e densità-indipendenti. Questo metodo efficace di etichettatura e specifica consente l&#39;arricchimento di 5-HMC con fondo estremamente basso e high-throughput mappatura epigenomic tramite sequenziamento di prossima generazione.

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 ...

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11.9K Views.  Emory University School of Medicine. Descritto è un processo a due fasi usando etichettatura β-glucosil (β-GT) per trasferire una azide-glucosio al 5-HMC, seguita dalla chimica click per trasferire un linker biotina per l'arricchimento facile e densità-indipendenti. Questo metodo efficace di etichettatura e specifica consente l'arricchimento di 5-HMC con fondo estremamente basso e high-throughput mappatura epigenomic tramite sequenziamento di prossima generazione.

Video: Selective Capture of 5-hydroxymethylcytosine from Genomic DNA

Isolation of Genomic DNA from Mouse Tails

Isolamento del DNA genomico da Tails mouse

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Biologia

Large Insert Environmental Genomic Library Production

The vast majority of microbes in nature currently remain inaccessible to traditional cultivation methods. Over the past decade, culture-independent environmental genomic (i.e. metagenomic) approaches have emerged, enabling researchers to bridge this cultivation gap by capturing the genetic content of indigenous microbial communities directly from the environment. To this end, genomic DNA libraries are constructed using standard albeit artful laboratory cloning techniques. Here we describe the construction of a large insert environmental genomic fosmid library with DNA derived from the vertical depth continuum of a seasonally hypoxic fjord. This protocol is directly linked to a series of connected protocols including coastal marine water sampling [1], large volume filtration of microbial biomass [2] and a DNA extraction and purification protocol [3]. At the outset, high quality genomic DNA is end-repaired with the creation of 5 -phosphorylated blunt ends. End-repaired DNA is subjected to pulsed-field gel electrophoresis (PFGE) for size selection and gel extraction is performed to recover DNA fragments between 30 and 60 thousand base pairs (Kb) in length. Size selected DNA is purified away from the PFGE gel matrix and ligated to the phosphatase-treated blunt-end fosmid CopyControl vector pCC1 (EPICENTRE <a href="http://www.epibio.com/item.asp?ID=385">http://www.epibio.com/item.asp?ID=385</a>). Linear concatemers of pCC1 and insert DNA are subsequently headfull packaged into phage particles by lambda terminase, with subsequent infection of phage-resistant E. coli cells. Successfully transduced clones are recovered on LB agar plates under antibiotic selection and archived in 384-well plate format using an automated colony picking robot (Qpix2, GENETIX). The current protocol draws from various sources including the CopyControl Fosmid Library Production Kit from EPICENTRE and the published works of multiple research groups [4-7]. Each step is presented with best practice in mind. Whenever possible we highlight subtleties in execution to improve overall quality and efficiency of library production. The whole process of fosmid library production and automated colony picking takes at least 7-10 days as there are many incubation steps included. However, there are several stopping points possible which are mentioned within the protocol.

Construction of a fosmid library with environmental genomic DNA isolated from the vertical depth continuum of a seasonally hypoxic fjord is described. The resulting clone library is picked into 384-well plates and archived for downstream sequencing and functional screening by the application of an automated colony picking system.

Inserire grande Ambientale Genomic Produzione Biblioteca

The vast majority of microbes in nature currently remain inaccessible to traditional cultivation methods. Over the past decade, culture-independent ...

Extraction of High Molecular Weight Genomic DNA from Soils and Sediments

The soil microbiome is a vast and relatively unexplored reservoir of genomic diversity and metabolic innovation that is intimately associated with nutrient and energy flow within terrestrial ecosystems. Cultivation-independent environmental genomic, also known as metagenomic, approaches promise unprecedented access to this genetic information with respect to pathway reconstruction and functional screening for high value therapeutic and biomass conversion processes. However, the soil microbiome still remains a challenge largely due to the difficulty in obtaining high molecular weight DNA of sufficient quality for large insert library production. Here we introduce a protocol for extracting high molecular weight, microbial community genomic DNA from soils and sediments. The quality of isolated genomic DNA is ideal for constructing large insert environmental genomic libraries for downstream sequencing and screening applications. 
The procedure starts with cell lysis. Cell walls and membranes of microbes are lysed by both mechanical (grinding) and chemical forces (&beta;-mercaptoethanol). Genomic DNA is then isolated using extraction buffer, chloroform-isoamyl alcohol and isopropyl alcohol. The buffers employed for the lysis and extraction steps include guanidine isothiocyanate and hexadecyltrimethylammonium bromide (CTAB) to preserve the integrity of the high molecular weight genomic DNA. Depending on your downstream application, the isolated genomic DNA can be further purified using cesium chloride (CsCl) gradient ultracentrifugation, which reduces impurities including humic acids. The first procedure, extraction, takes approximately 8 hours, excluding DNA quantification step. The CsCl gradient ultracentrifugation, is a two days process. During the entire procedure, genomic DNA should be treated gently to prevent shearing, avoid severe vortexing, and repetitive harsh pipetting.

A methodology to isolate high molecular weight and high quality genomic DNA from soil microbial community is described.

Estrazione di DNA ad alta peso molecolare genomico da terreni e sedimenti

The soil microbiome is a vast and relatively unexplored reservoir of genomic diversity and metabolic innovation that is intimately associated with ...

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

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 ancient bones. The method greatly reduces sample destruction and sequencing demands relative to direct PCR or shotgun sequencing approaches. We used this method to reconstruct the complete mitochondrial DNA (mtDNA) genomes of five Neandertals from across their geographic range. The mtDNA genetic diversity of the late Neandertals was approximately three times lower than that of contemporary modern humans. Together with analyses of mtDNA protein evolution, these data suggest that the long-term effective population size of Neandertals was smaller than that of modern humans and extant great apes.

We present a method of targeted ancient DNA sequence retrieval, which we used to reconstruct the complete mitochondrial genomes of five Neandertal individuals. Comparison of these sequences with present day humans suggests that Neandertals had a long term low effective population size.

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 ...

Generation of RNA/DNA Hybrids in Genomic DNA by Transformation using RNA-containing Oligonucleotides

Synthetic short nucleic acid polymers, oligonucleotides (oligos), are the most functional and widespread tools of molecular biology. Oligos can be produced to contain any desired DNA or RNA sequence and can be prepared to include a wide variety of base and sugar modifications. Moreover, oligos can be designed to mimic specific nucleic acid alterations and thus, can serve as important tools to investigate effects of DNA damage and mechanisms of repair. We found that Thermo Scientific Dharmacon RNA-containing oligos with a length between 50 and 80 nucleotides can be particularly suitable to study, in vivo, functions and consequences of chromosomal RNA/DNA hybrids and of ribonucleotides embedded into DNA. RNA/DNA hybrids can readily form during DNA replication, repair and transcription, however, very little is known about the stability of RNA/DNA hybrids in cells and to which extent these hybrids can affect the genetic integrity of cells. RNA-containing oligos, therefore, represent a perfect vector to introduce ribonucleotides into chromosomal DNA and generate RNA/DNA hybrids of chosen length and base composition. Here we present the protocol for the incorporation of ribonucleotides into the genome of the eukaryotic model system yeast /Saccharomyces cerevisiae/. Yet, our lab has utilized Thermo Scientific Dharmacon RNA-containing oligos to generate RNA/DNA hybrids at the chromosomal level in different cell systems, from bacteria to human cells.

This work shows how to form an RNA/DNA hybrid at the chromosomal level and reveal transfer of genetic information from RNA to genomic DNA in yeast cells.

Generazione di DNA / RNA ibrido nel DNA genomico da trasformazione utilizzando RNA contenenti oligonucleotidi

Synthetic short nucleic acid polymers, oligonucleotides (oligos), are the most functional and widespread tools of molecular biology. Oligos can be ...

Streamlined Purification of Plasmid DNA From Prokaryotic Cultures

We describe the complete process of AcroPrep Advance Filter Plates for 96 plasmid preparations, starting from prokaryotic culture and ending with high purity DNA. Based on multi-well filtration for bacterial lysate clearance and DNA purification, this method creates a streamlined process for plasmid preparation. Filter plates containing silica-based media can easily be processed by vacuum filtration or centrifuge to yield appreciable quantities of plasmid DNA. Quantitative analyses determine the purified plasmid DNA is consistently of high quality with average OD260/280 ratios of 1.97. Overall, plasmid yields offer more pure DNA for downstream applications, such as sequencing and cloning. This streamlined method of using AcroPrep Advance Filter Plates allows for manual, semi-automated or fully-automated processing.

This protocol is a cost effective alternative for efficient parallel clarification and plasmid DNA purification from E. coli cultures. The AcroPrep Advance process starts with an optimized lysate clarification filter plate followed by purification on a high binding capacity DNA binding filter plate.

Purificazione semplificata del DNA plasmidi da culture procariotiche

We describe the complete process of AcroPrep Advance Filter Plates for 96 plasmid preparations, starting from prokaryotic culture and ending with high ...

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 ...

Competitive Genomic Screens of Barcoded Yeast Libraries

By virtue of advances in next generation sequencing technologies, we have access to new genome sequences almost daily. The tempo of these advances is accelerating, promising greater depth and breadth. In light of these extraordinary advances, the need for fast, parallel methods to define gene function becomes ever more important. Collections of genome-wide deletion mutants in yeasts and E. coli have served as workhorses for functional characterization of gene function, but this approach is not scalable, current gene-deletion approaches require each of the thousands of genes that comprise a genome to be deleted and verified. Only after this work is complete can we pursue high-throughput phenotyping. Over the past decade, our laboratory has refined a portfolio of competitive, miniaturized, high-throughput genome-wide assays that can be performed in parallel. This parallelization is possible because of the inclusion of DNA 'tags', or 'barcodes,' into each mutant, with the barcode serving as a proxy for the mutation and one can measure the barcode abundance to assess mutant fitness. In this study, we seek to fill the gap between DNA sequence and barcoded mutant collections. To accomplish this we introduce a combined transposon disruption-barcoding approach that opens up parallel barcode assays to newly sequenced, but poorly characterized microbes. To illustrate this approach we present a new Candida albicans barcoded disruption collection and describe how both microarray-based and next generation sequencing-based platforms can be used to collect 10,000 - 1,000,000 gene-gene and drug-gene interactions in a single experiment.

We have developed comprehensive, unbiased genome-wide screens to understand gene-drug and gene-environment interactions. Methods for screening these mutant collections are presented.

Schermi competitivo genomica delle Biblioteche di lievito con codice a barre

By virtue of advances in next generation sequencing technologies, we have access to new genome sequences almost daily.  The tempo of these advances is ...

Extraction of High Molecular Weight DNA from Microbial Mats

Successful and accurate analysis and interpretation of metagenomic data is dependent upon the efficient extraction of high-quality, high molecular weight (HMW) community DNA. However, environmental mat samples often pose difficulties to obtaining large concentrations of high-quality, HMW DNA. Hypersaline microbial mats contain high amounts of extracellular polymeric substances (EPS)1 and salts that may inhibit downstream applications of extracted DNA. Direct and harsh methods are often used in DNA extraction from refractory samples. These methods are typically used because the EPS in mats, an adhesive matrix, binds DNA2,3 during direct lysis. As a result of harsher extraction methods, DNA becomes fragmented into small sizes4,5,6.
The DNA thus becomes inappropriate for large-insert vector cloning. In order to circumvent these limitations, we report an improved methodology to extract HMW DNA of good quality and quantity from hypersaline microbial mats. We employed an indirect method involving the separation of microbial cells from the background mat matrix through blending and differential centrifugation. A combination of mechanical and chemical procedures was used to extract and purify DNA from the extracted microbial cells. Our protocol yields approximately 2 &mu;g of HMW DNA (35-50 kb) per gram of mat sample, with an A260/280 ratio of 1.6. Furthermore, amplification of 16S rRNA genes7 suggests that the protocol is able to minimize or eliminate any inhibitory effects of contaminants. Our results provide an appropriate methodology for the extraction of HMW DNA from microbial mats for functional metagenomic studies and may be applicable to other environmental samples from which DNA extraction is challenging.

We provide an improved protocol for extracting high molecular weight DNA from hypersaline microbial mats. Microbial cells are separated from the mat matrix prior to DNA extraction and purification. This enhances the concentrations, quality, and size of the DNA. The protocol may be used for other refractory samples.

Estrazione di DNA ad alta peso molecolare da Mats microbica

Successful and accurate analysis and interpretation of metagenomic data is dependent upon the efficient extraction  of high-quality, high molecular weight ...

Detection of Rare Genomic Variants from Pooled Sequencing Using SPLINTER

As DNA sequencing technology has markedly advanced in recent years2, it has become increasingly evident that the amount of genetic variation between any two individuals is greater than previously thought3. In contrast, array-based genotyping has failed to identify a significant contribution of common sequence variants to the phenotypic variability of common disease4,5. Taken together, these observations have led to the evolution of the Common Disease / Rare Variant hypothesis suggesting that the majority of the "missing heritability" in common and complex phenotypes is instead due to an individual's personal profile of rare or private DNA variants6-8. However, characterizing how rare variation impacts complex phenotypes requires the analysis of many affected individuals at many genomic loci, and is ideally compared to a similar survey in an unaffected cohort. Despite the sequencing power offered by today's platforms, a population-based survey of many genomic loci and the subsequent computational analysis required remains prohibitive for many investigators.
To address this need, we have developed a pooled sequencing approach1,9 and a novel software package1 for highly accurate rare variant detection from the resulting data. The ability to pool genomes from entire populations of affected individuals and survey the degree of genetic variation at multiple targeted regions in a single sequencing library provides excellent cost and time savings to traditional single-sample sequencing methodology. With a mean sequencing coverage per allele of 25-fold, our custom algorithm, SPLINTER, uses an internal variant calling control strategy to call insertions, deletions and substitutions up to four base pairs in length with high sensitivity and specificity from pools of up to 1 mutant allele in 500 individuals. Here we describe the method for preparing the pooled sequencing library followed by step-by-step instructions on how to use the SPLINTER package for pooled sequencing analysis (<a href="http://www.ibridgenetwork.org/wustl/splinter" target="_blank">http://www.ibridgenetwork.org/wustl/splinter</a>). We show a comparison between pooled sequencing of 947 individuals, all of whom also underwent genome-wide array, at over 20kb of sequencing per person. Concordance between genotyping of tagged and novel variants called in the pooled sample were excellent. This method can be easily scaled up to any number of genomic loci and any number of individuals. By incorporating the internal positive and negative amplicon controls at ratios that mimic the population under study, the algorithm can be calibrated for optimal performance. This strategy can also be modified for use with hybridization capture or individual-specific barcodes and can be applied to the sequencing of naturally heterogeneous samples, such as tumor DNA.

Pooled DNA sequencing is a fast and cost-effective strategy to detect rare variants associated with complex phenotypes in large cohorts. Here we describe the computational analysis of pooled, next-generation sequencing of 32 cancer-related genes using the SPLINTER software package. This method is scalable, and applicable to any phenotype of interest.

Rilevamento di Rare varianti genomiche da Sequencing pool Uso SPLINTER

As DNA sequencing technology has markedly advanced in recent years2, it has become increasingly evident that the amount of genetic variation between any ...