This protocol development was supported by the Bill and Melinda Gates Foundation Grand Challenges in Global Health grant 37774. Real-time PCR reagents and advice were provided by Abbott Molecular Inc. Des Plaines, IL. 8E5-LAV cells were provided by the Virology Quality Assurance Laboratory, Rush Presbyterian; St. Luke's Medical Center. HIV-1 negative blood was provided by Core Lab, NorthShore University HealthSystems, Evanston, IL. Thanks to Mark Fisher for photography help.

Etica dichiarazione: I campioni di sangue intero utilizzate in questo studio non sono considerati come ricerca che coinvolge soggetti umani. I campioni sono stati ottenuti per scopi diagnostici clinici, che sono state soddisfatte, e la porzione restante di questi campioni è stato fornito per il dosaggio di ricerca FINA. I campioni sono stati codificati in modo tale che i ricercatori non sono stati in grado di accertare facilmente l&#39;identità degli individui. 1. Preparazione della FINA campione Modulo di preparazione <ol><li> Preparare tampone assorbente tagliando un 35 x 35 mm 2 di un 2,6 mm batuffolo di cotone al 100% di spessore. Tagliare una pastiglia per ogni campione. </li><li> Preparare parafilm plastica con un nastro di protezione di carta da un pezzo di taglio della pellicola di paraffina (25 mm (h) di 50 mm (w)) e quindi un foro di diametro 7,14 millimetri al centro del nastro con un martello guidato perforazione. Preparare un nastro per ogni campione. </li><li> Preparare un separatore membrana di sangue vetro rilegato (membrana cattura) disco perforando un cerchio di diametro 8,35 millimetricon un martello guidato perforazione. Punch un disco per ogni campione. Il disco ha una capacità di ritenzione delle particelle di 2,3 micron. </li><li> Assemblare il modulo di preparazione del campione FINA disponendo il disc cattura nel centro del pad blotting utilizzando pinze. Posizionare il nastro parafilm sopra il disco acquisizione in modo che il foro del nastro paraffina lascia il centro del disco cattura esposto. </li><li> Garantire il contatto tra il disco e il tampone assorbente allungando il nastro di paraffina leggermente a coprire il pad assorbente e premendo saldamente il nastro di paraffina a bastone per il pad assorbente lungo tutti i bordi del disco. </li><li> Fare come molti moduli come necessario per l&#39;esperimento o di riserva per un utilizzo futuro. </li></ol> 2. Preparazione della qPCR tubo <ol><li> Preparare un disco nastro 5,1 millimetri che ancorare la membrana cattura cella a lato del tubo qPCR per impedire la membrana blocchi la rilevazione della fluorescenza per punzonatura un cerchio di polie doppio rivestimentoster nastro diagnostico con un martello guidato pugno da un foglio di nastro. Preparare un disco a nastro per ogni campione. </li><li> Rimuovere un lato del rivestimento adesivo del nastro. Posizionare il nastro in 200 microlitri tubo PCR, attaccare su un lato e verso il fondo della provetta. Rimuovere il secondo rivestimento adesivo. Il tubo è ora pronto a ricevere disco preparato. </li><li> Produrre come molti tubi come necessario per l&#39;esperimento o scorte per uso futuro. </li></ol> 3. Contrive sangue del campione Nota: Se si sta preparando un vero e proprio provino, passare al punto 4. <ol><li> Scongelare le cellule 8e5-LAV 15 portatori di una singola copia dei provirus HIV-1 ottenuti dal Laboratorio Quality Assurance Virology (VQA; Rush Presbyterian / St Luke Medical Center, Chicago, IL.). Nota: Vengono memorizzati come pellet cellulari congelati ad una concentrazione di 4.000 cellule / microlitro. Conteggio delle cellule è stata verificata come descritto in precedenza 9. </li><li> preparare serdiluizione ial a congelamento medio (siero 90% fetale bovino, 10% dimetilsolfossido) a concentrazioni che vanno da 1 a 400 cellule / microlitro. </li><li> Pipettare 10 celle microlitri di ciascuna delle diluizioni seriali in una provetta. Aggiungere 100 ml di HIV-1 negativo EDTA campione di sangue intero fresco trattati ad ogni provetta per creare una curva standard di 8e5-LAV numero di copie 10-4,000. Mescolare delicatamente sfogliando il fondo del tubo 5 volte. </li></ol> 4. Eseguire FINA Estrazione <ol><li> globuli Lyse aggiungendo Triton-X100) ad una concentrazione finale di 1% (11 microlitri 10% Triton-X100 a 110 microlitri di sangue intero e cellule dal punto 3.3). </li><li> Mescolare delicatamente sfogliando il fondo del tubo 5 volte. Il sangue dovrebbe diventare rosso traslucido. </li><li> Pipettare tutti i campioni di sangue sul disco cattura. Nota: Una guarnizione a tenuta d&#39;acqua tra il nastro paraffina e la membrana cattura assicura che il sangue scorre attraverso la membrana, e un buon contatto tra l&#39;acquisizione membrane e assorbente complesso a tampone garantisce un rapido assorbimento del campione. </li><li> Lasciare in ammollo campione attraverso il filtro. Nota: Gli stoppini lisato sangue nel tampone assorbente per capillarità, catturando il DNA genomico sulla superficie del disco cattura. Il lisato è imbevuto nel disco quando appare opaca. </li><li> Aggiungere 600-1,000 ml 10 mM di NaOH goccia a goccia sul disco cattura. Nota: Il tampone di lavaggio può anche essere aggiunto come una massa aggiunta di 600 microlitri. Il buffer formerà una gocciolina sul nastro di paraffina idrofoba e stoppino attraverso il foro sul disco in circa 20 sec. Il filtro passa dal rosso al bianco indicando liquidazione di emoglobina. </li><li> Separare il disco dal tampone assorbente con una pinza e applicare il disco al nastro nel tubo PCR preparato. Se c&#39;è un colore rosso rimasto sul filtro aggiungere 50-100 ml di tampone di lavaggio per cancellare il filtro prima di mettere nel tubo. Nota: Raramente, l&#39;eccesso di pressione applicata durante la preparazione dila FINA moduli comporta il partizionamento degli strati di membrana durante la separazione dal tampone assorbente. Eliminare questi dischi e preparare un campione fresco. </li><li> Dopo il filtro è posizionato nel tubo PCR, analizzare i campioni subito. In alternativa, asciugare per una notte in una scatola contenente solfato di calcio essiccante, quindi cap e mettere in un sacchetto di alluminio con essiccante di silice e conservare fino al momento dell&#39;analisi. </li></ol> 5. qPCR Reazione <ol><li> Preparare 100 microlitri qPCR master mix per reazione con l&#39;HIV primer e sonde specifiche come descritto in precedenza 9,11. Includere un controllo interno di amplificazione per monitorare la presenza di inibitori di amplificazione e di verificare che un campione negativo è un vero negativo. Assicurarsi che il filtro è completamente ricoperta di master mix e che il filtro rimane fissato al lato del tubo durante la reazione. </li><li> Eseguire l&#39;amplificazione utilizzando un dispositivo di PCR in tempo reale, come descritto in precedenza 9. </li></ol>

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The authors have nothing to disclose.

EID legata al trattamento di accesso rapido ha dimostrato di ridurre la mortalità infantile a causa di infezione da HIV 16. A causa della persistenza di anticorpi materni nel sangue di un neonato, rapidi test degli anticorpi HIV hanno limitato l&#39;utilità per determinare il carattere dei bambini HIV-esposti. L&#39;OMS raccomanda che tutti i bambini nati da madri HIV-1 positivi dovrebbero essere testati a 4-6 settimane di età, utilizzando un test virologico 17. Abbiamo riportato lo sviluppo di ...

Diversi rapporti hanno discusso lo sviluppo di metodi di estrazione carta- o membrana a base per l&#39;uso in point-of-care (POC) dispositivi di 1-5, con l&#39;obiettivo di rendere la sensibilità squisita e la specificità della diagnostica molecolare a disposizione di tutti. L&#39;Organizzazione Mondiale della Sanità (OMS) Malattie Sessualmente Trasmesse Diagnostica iniziativa ha coniato il termine ASSURED (a prezzi accessibili, sensibile, specifico, facile da usare, veloce e robusto, senza attrezzature e distribuito chi ne ha bisogno) per descrivere le caratteristiche ideali di un POC Test 6. Di queste linee guida, la caratteristica senza attrezzatura è particolarmente difficile da raggiungere per la diagnostica molecolare. Tuttavia, ogni innovazione nel campo avanzerà l&#39;obiettivo di raggiungere le più bisognose, e non c&#39;è speranza di miglioramento a breve termine in termini di prestazioni di prova adattando la tecnologia esistente 7. Qui si descrive un protocollo semplice per l&#39;estrazione del DNA da sangue interoche non richiede chimica complessa o strumentazione di laboratorio. La FINA (isolamento filtrazione di acidi nucleici) metodo di preparazione campione è stato originariamente sviluppato per estrarre il DNA dei leucociti dal sangue intero per rilevare l&#39;HIV-1 provirus come parte di un point-of-care (POC) del campione alla risposta PCR quantitativa diagnostica piattaforma (EID) (qPCR) precoce infantile per l&#39;utilizzo in contesti di risorse limitate 8-11. Estrazione FINA differisce dai metodi di purificazione convenzionali che utilizzano membrane di silice o silice particelle paramagnetiche rivestite di legarsi reversibilmente DNA in presenza di agenti caotropici 12. Invece, FINA utilizza filtrazione verticale attraverso una membrana di separazione per estrarre DNA cellulare da sangue intero direttamente. La membrana contenente il DNA intrappolata può essere collocato direttamente in un tubo di PCR e sia immediatamente utilizzato in una reazione PCR o aria secca per la successiva amplificazione 9. Senza caotropici agenti, fenolo, o alcoli sono utilizzati per l&#39;estrazione del campione, eliminaTing le ampie fasi di lavaggio necessari per rimuovere potenti inibitori qPCR derivanti dal processo di estrazione 13,14. La membrana FINA può catturare sia cellule 9 o DNA genomico liberati da lisi cellulare 11 prima che il campione viene aggiunto alla membrana. Per la cattura delle cellule, sangue intero viene aggiunto direttamente alla membrana. Le cellule sono poi lisate nella membrana per aggiunta di 10 mM NaOH. Il vantaggio di questo metodo è che si tratta solo 3 fasi: 1) l&#39;aggiunta del campione; 2) la lisi cellulare / lavaggio e 3) il posizionamento del disco filtro in tubo qPCR. Lo svantaggio di questo metodo è che la membrana può contenere solo un numero definito di cellule direttamente proporzionale al diametro del disco membrana. Per raggiungere il limite di rilevazione necessario per EID, 100 ml di sangue intero è richiesto per l&#39;ingresso di campionamento che comporta un filtro che è troppo grande per essere inserito in un tubo qPCR. Lisi delle cellule del sangue con detersivo prima di aggiungere il campione alla raccoltamembrana aggiunge una fase di elaborazione, ma consente l&#39;uso di un filtro più piccolo per la stessa dimensione del campione. Siamo stati in grado di dimostrare alta riproducibilità, la rilevazione solo esemplare, e la quantificazione di un minimo di 10 copie di HIV-1 DNA provirale da 100 ml di sangue che utilizzano questa configurazione di prova 11. In questo rapporto, descriviamo il protocollo FINA come originariamente sviluppato per l&#39;uso in laboratorio. Il sandwich membrana / filtro noto come modulo di preparazione del campione FINA può essere preparato in grandi lotti e stoccato per un uso successivo. Quando i campioni vengono estratti questo processo richiede 2 min e può essere eseguita in diversi lotti di dimensioni. La qPCR può essere eseguito immediatamente o dei filtri contenenti il ​​DNA incorporato può essere conservato fino a quando non è conveniente per eseguire qPCR. Questo metodo è molto comodo per le analisi di routine, di esemplari in entrambe le impostazioni alte e basse risorse.

<table border="0" cellpadding="0" cellspacing="0" width="860">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:432px;">Fusion 5</td>
			<td style="width:179px;">GE Healthcare Life Sciences</td>
			<td style="width:104px;">8151-9915</td>
			<td style="width:147px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">707 blotting pad</td>
			<td>VWR International</td>
			<td>28298-014</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PARAFILM M</td>
			<td>VWR International</td>
			<td>52858-000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">3M Double-Coated Polyester Diagnostic Tape&#160;</td>
			<td>3M Medical Specialties</td>
			<td>9965</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">200 &#181;l qPCR strip tubes</td>
			<td>Agilent</td>
			<td>401428</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">optical strip caps</td>
			<td>Agilent</td>
			<td>401425</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mx3005p qPCR System</td>
			<td>Agilent</td>
			<td>401456</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">sodium hydroxide</td>
			<td>Sigma-Aldrich</td>
			<td>221465</td>
			<td>A.C.S. Reagent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Triton X-100</td>
			<td>Sigma-Aldrich</td>
			<td>T9284</td>
			<td>BioXtra</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">dimethyl sulfoxide</td>
			<td>Sigma-Aldrich</td>
			<td>D8418</td>
			<td>for molecular biology</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">fetal bovine serum, certified, U.S. origin</td>
			<td>Thermo Fisher Scientific</td>
			<td>16000-044</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hammer driven small hole punch 3/16&#34; hole diameter (5.1 mm)</td>
			<td>McMaster Carr</td>
			<td>3424A16</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hammer driven small hole punch 1/4&#34; hole diameter (7.14 mm)</td>
			<td>McMaster Carr</td>
			<td>3424A19</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hammer driven small hole punch 5/16&#34; hole diameter (8.35 mm)</td>
			<td>McMaster Carr</td>
			<td>3424A23</td>
			<td></td>
		</tr>
	</tbody>
</table>

filtration isolation of nucleic acids:  a simple and rapid dna extraction method

FINA, isolamento filtrazione di acidi nucleici, è un metodo di estrazione romanzo che utilizza filtrazione verticale tramite una membrana di separazione e tampone assorbente per estrarre DNA cellulare da sangue intero in meno di 2 min. Il campione di sangue viene trattata con un detergente, brevemente mescolato e applicato dalla pipetta alla membrana di separazione. Il lisato stoppini nel tampone assorbente per capillarità, catturando il DNA genomico sulla superficie della membrana di separazione. Il DNA estratto viene conservato sulla membrana nel corso di una semplice fase di lavaggio in cui gli inibitori della PCR sono malvagi nel tampone assorbente assorbente. La membrana contenente il DNA intrappolata viene quindi aggiunto alla reazione PCR senza ulteriore purificazione. Questo semplice metodo non richiede attrezzature di laboratorio e può essere facilmente implementato con materiale di laboratorio poco costoso. Qui si descrive un protocollo per il rilevamento ad alta sensibilità e la quantificazione del virus HIV-1 DNA provirale da 100 ml di sangue intero come modello per l&#39;inizio inDiagnosi fant del virus HIV che potrebbe facilmente essere adattato per altri obiettivi genetici.

Il flusso di lavoro per l&#39;estrazione del DNA provirale dal sangue intero addizionato di cellule 8e5-LAV è mostrato in Figura 1. La Figura 2 mostra il modulo di esempio FINA e preparati tubo qPCR. Questo metodo consente efficiente amplificazione del HIV-1 provirus da cellule 8e5-LAV a differenti numeri di copia, come mostrato nella curva standard di esemplari artificiosa (Figura 3). PCR ha avuto un rendimento del 103%, come calcolato...

Watch this Scientific Journal Video about Filtration Isolation of Nucleic Acids:  A Simple and Rapid DNA Extraction Method at JoVE.com

Filtration Isolation of Nucleic Acids:  A Simple and Rapid DNA Extraction Method

Descriviamo qui un metodo di estrazione del DNA semplice e rapida cartaceo di HIV provirale DNA da sangue intero rilevato mediante PCR quantitativa. Questo protocollo può essere esteso per l&#39;utilizzo nel rilevare altri marcatori genetici o utilizzando metodi di amplificazione alternativi.

L&#39;isolamento di filtrazione di acidi nucleici: DNA metodo di estrazione semplice e veloce

FINA, filtration isolation of nucleic acids, is a novel extraction method which utilizes vertical filtration via a separation membrane and absorbent pad ...

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Biology

18.0K Views.  Northwestern University.  Descriviamo qui un metodo di estrazione del DNA semplice e rapida cartaceo di HIV provirale DNA da sangue intero rilevato mediante PCR quantitativa. Questo protocollo può essere esteso per l'utilizzo nel rilevare altri marcatori genetici o utilizzando metodi di amplificazione alternativi. 

Video: Filtration Isolation of Nucleic Acids:  A Simple and Rapid DNA Extraction Method

NanoDrop Microvolume Quantitation of Nucleic Acids

Biomolecular assays are continually being developed that use progressively smaller amounts of material, often precluding the use of conventional cuvette-based instruments for nucleic acid quantitation for those that can perform microvolume quantitation.
The NanoDrop microvolume sample retention system (Thermo Scientific NanoDrop Products) functions by combining fiber optic technology and natural surface tension properties to capture and retain minute amounts of sample independent of traditional containment apparatus such as cuvettes or capillaries. Furthermore, the system employs shorter path lengths, which result in a broad range of nucleic acid concentration measurements, essentially eliminating the need to perform dilutions. Reducing the volume of sample required for spectroscopic analysis also facilitates the inclusion of additional quality control steps throughout many molecular workflows, increasing efficiency and ultimately leading to greater confidence in downstream results.
The need for high-sensitivity fluorescent analysis of limited mass has also emerged with recent experimental advances. Using the same microvolume sample retention technology, fluorescent measurements may be performed with 2 &mu;L of material, allowing fluorescent assays volume requirements to be significantly reduced. Such microreactions of 10 &mu;L or less are now possible using a dedicated microvolume fluorospectrometer.
Two microvolume nucleic acid quantitation protocols will be demonstrated that use integrated sample retention systems as practical alternatives to traditional cuvette-based protocols. First, a direct A260 absorbance method using a microvolume spectrophotometer is described. This is followed by a demonstration of a fluorescence-based method that enables reduced-volume fluorescence reactions with a microvolume fluorospectrometer. These novel techniques enable the assessment of nucleic acid concentrations ranging from 1 pg/ &mu;L to 15,000 ng/ &mu;L with minimal consumption of sample.

The use of NanoDrop microvolume systems as practical and efficient alternatives to traditional nucleic acid quantitation methodology is described through the demonstration of two microvolume nucleic acid quantitation protocols.

NanoDrop microvolumi quantificazione degli acidi nucleici

Biomolecular assays are continually being developed that use progressively smaller amounts of material, often precluding the use of conventional ...

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Biologia

Concentration Determination of Nucleic Acids and Proteins Using the Micro-volume Bio-spec Nano Spectrophotometer

Nucleic Acid quantitation procedures have advanced significantly in the last three decades. More and more, molecular biologists require consistent small-volume analysis of nucleic acid samples for their experiments. The BioSpec-nano provides a potential solution to the problems of inaccurate, non-reproducible results, inherent in current DNA quantitation methods, via specialized optics and a sensitive PDA detector. The BioSpec-nano also has automated functionality such that mounting, measurement, and cleaning are done by the instrument, thereby eliminating tedious, repetitive, and inconsistent placement of the fiber optic element and manual cleaning.
In this study, data is presented on the quantification of DNA and protein, as well as on measurement reproducibility and accuracy. Automated sample contact and rapid scanning allows measurement in three seconds, resulting in excellent throughput. Data analysis is carried out using the built-in features of the software. The formula used for calculating DNA concentration is:
Sample Concentration = DF &middot; (OD260-OD320)&middot; NACF (1)
Where DF = sample dilution factor and NACF = nucleic acid concentration factor.
The Nucleic Acid concentration factor is set in accordance with the analyte selected1.
Protein concentration results can be expressed as &mu;g/ mL or as moles/L by entering e280 and molecular weight values respectively. When residue values for Tyr, Trp and Cysteine (S-S bond) are entered in the e280Calc tab, the extinction coefficient values are calculated as e280 = 5500 x (Trp residues) + 1490 x (Tyr residues) + 125 x (cysteine S-S bond). The e280 value is used by the software for concentration calculation.
In addition to concentration determination of nucleic acids and protein, the BioSpec-nano can be used as an ultra micro-volume spectrophotometer for many other analytes or as a standard spectrophotometer using 5 mm pathlength cells.

This communication presents data on the accuracy and reproducibility of the BioSpec-nano UV-VIS spectrophotometer for dsDNA and protein quantitation. Even with ultra-small volumes (1 to 2 L), reproducibility is excellent, while the automated wiping function improves throughput and results in minimal carryover for more precise results.

Determinazione concentrazione di acidi nucleici e proteine ​​Utilizzando il Micro-volume Bio-spec Spettrofotometro Nano

Nucleic Acid quantitation procedures have advanced significantly in the last three decades.  More and more, molecular biologists require consistent ...

DNA Extraction from Paraffin Embedded Material for Genetic and Epigenetic Analyses

Disease development and progression are characterized by frequent genetic and epigenetic aberrations including chromosomal rearrangements, copy number gains and losses and DNA methylation. Advances in high-throughput, genome-wide profiling technologies, such as microarrays, have significantly improved our ability to identify and detect these specific alterations. However as technology continues to improve, a limiting factor remains sample quality and availability. Furthermore, follow-up clinical information and disease outcome are often collected years after the initial specimen collection. Specimens, typically formalin-fixed and paraffin embedded (FFPE), are stored in hospital archives for years to decades. DNA can be efficiently and effectively recovered from paraffin-embedded specimens if the appropriate method of extraction is applied. High quality DNA extracted from properly preserved and stored specimens can support quantitative assays for comparisons of normal and diseased tissues and generation of genetic and epigenetic signatures 1. To extract DNA from paraffin-embedded samples, tissue cores or microdissected tissue are subjected to xylene treatment, which dissolves the paraffin from the tissue, and then rehydrated using a series of ethanol washes. Proteins and harmful enzymes such as nucleases are subsequently digested by proteinase K. The addition of lysis buffer, which contains denaturing agents such as sodium dodecyl sulfate (SDS), facilitates digestion 2. Nucleic acids are purified from the tissue lysate using buffer-saturated phenol and high speed centrifugation which generates a biphasic solution. DNA and RNA remain in the upper aqueous phase, while proteins, lipids and polysaccharides are sequestered in the inter- and organic-phases respectively. Retention of the aqueous phase and repeated phenol extractions generates a clean sample. Following phenol extractions, RNase A is added to eliminate contaminating RNA. Additional phenol extractions following incubation with RNase A are used to remove any remaining enzyme. The addition of sodium acetate and isopropanol precipitates DNA, and high speed centrifugation is used to pellet the DNA and facilitate isopropanol removal. Excess salts carried over from precipitation can interfere with subsequent enzymatic assays, but can be removed from the DNA by washing with 70% ethanol, followed by centrifugation to re-pellet the DNA 3. DNA is re-suspended in distilled water or the buffer of choice, quantified and stored at -20&deg;C. Purified DNA can subsequently be used in downstream applications which include, but are not limited to, PCR, array comparative genomic hybridization 4 (array CGH), methylated DNA Immunoprecipitation (MeDIP) and sequencing, allowing for an integrative analysis of tissue/tumor samples.

This video demonstrates the protocol for DNA extraction from formalin-fixed paraffin-embedded material. This is a multi-day procedure in which tissue sections are deparaffinized with xylene, rehydrated with ethanol and treated with proteinase K to purify and isolate DNA for subsequent gene-specific or genome-wide analysis.

Estrazione DNA da materiale in paraffina per le analisi genetiche ed epigenetiche

Disease development and progression are characterized by frequent genetic and epigenetic aberrations including chromosomal rearrangements, copy number ...

Electricity-Free, Sequential Nucleic Acid and Protein Isolation

Traditional and emerging pathogens such as Enterohemorrhagic Escherichia coli (EHEC), Yersinia pestis, or prion-based diseases are of significant concern for governments, industries and medical professionals worldwide. For example, EHECs, combined with Shigella, are responsible for the deaths of approximately 325,000 children each year and are particularly prevalent in the developing world where laboratory-based identification, common in the United States, is unavailable 1. The development and distribution of low cost, field-based, point-of-care tools to aid in the rapid identification and/or diagnosis of pathogens or disease markers could dramatically alter disease progression and patient prognosis. We have developed a tool to isolate nucleic acids and proteins from a sample by solid-phase extraction (SPE) without electricity or associated laboratory equipment 2. The isolated macromolecules can be used for diagnosis either in a forward lab or using field-based point-of-care platforms. Importantly, this method provides for the direct comparison of nucleic acid and protein data from an un-split sample, offering a confidence through corroboration of genomic and proteomic analysis.
Our isolation tool utilizes the industry standard for solid-phase nucleic acid isolation, the BOOM technology, which isolates nucleic acids from a chaotropic salt solution, usually guanidine isothiocyanate, through binding to silica-based particles or filters 3. CUBRC's proprietary solid-phase extraction chemistry is used to purify protein from chaotropic salt solutions, in this case, from the waste or flow-thru following nucleic acid isolation4.
By packaging well-characterized chemistries into a small, inexpensive and simple platform, we have generated a portable system for nucleic acid and protein extraction that can be performed under a variety of conditions. The isolated nucleic acids are stable and can be transported to a position where power is available for PCR amplification while the protein content can immediately be analyzed by hand held or other immunological-based assays. The rapid identification of disease markers in the field could significantly alter the patient's outcome by directing the proper course of treatment at an earlier stage of disease progression. The tool and method described are suitable for use with virtually any infectious agent and offer the user the redundancy of multi-macromolecule type analyses while simultaneously reducing their logistical burden.

A tool and chemistries are described to sequentially isolate nucleic acids followed by proteins from a sample without the need for electricity. The tool consists of a sorbent held within a transfer pipette while the isolation chemistries are based on solid-phase extraction principles. The isolated macromolecules can be analyzed by immuno-based and PCR-based assays.

Elettricità-Free, Nucleic Acid sequenziale e isolamento delle proteine

Traditional and emerging pathogens such as Enterohemorrhagic Escherichia coli (EHEC), Yersinia pestis, or prion-based diseases are of significant concern ...

Rapid Isolation And Purification Of Mitochondria For Transplantation By Tissue Dissociation And Differential Filtration

Previously described mitochondrial isolation methods using differential centrifugation and/or Ficoll gradient centrifugation require 60 to 100 min to complete. We describe a method for the rapid isolation of mitochondria from mammalian biopsies using a commercial tissue dissociator and differential filtration. In this protocol, manual homogenization is replaced with the tissue dissociator&#8217;s standardized homogenization cycle. This allows for uniform and consistent homogenization of tissue that is not easily achieved with manual homogenization. Following tissue dissociation, the homogenate is filtered through nylon mesh filters, which eliminate repetitive centrifugation steps. As a result, mitochondrial isolation can be performed in less than 30 min. This isolation protocol yields approximately 2 x 1010 viable and respiration competent mitochondria from 0.18 &#177; 0.04 g (wet weight) tissue sample.

A method for rapid isolation of mitochondria from mammalian tissue biopsies is described. Rat liver or skeletal muscle preparations were homogenized with a commercial tissue dissociator and mitochondria were isolated by differential filtration through nylon mesh filters. Mitochondrial isolation time is &#60;30 min compared to 60 - 100 min using alternative methods.

Isolamento rapida e purificazione di mitocondri per il trapianto da tessuto Dissociazione e Filtrazione Differenziale

Previously described mitochondrial isolation methods using differential centrifugation and/or Ficoll gradient centrifugation require 60 to 100 min to ...

Sequence-specific Labeling of Nucleic Acids and Proteins with Methyltransferases and Cofactor Analogues

S-Adenosyl-l-methionine (AdoMet or SAM)-dependent methyltransferases (MTase) catalyze the transfer of the activated methyl group from AdoMet to specific positions in DNA, RNA, proteins and small biomolecules. This natural methylation reaction can be expanded to a wide variety of alkylation reactions using synthetic cofactor analogues. Replacement of the reactive sulfonium center of AdoMet with an aziridine ring leads to cofactors which can be coupled with DNA by various DNA MTases. These aziridine cofactors can be equipped with reporter groups at different positions of the adenine moiety and used for Sequence-specific Methyltransferase-Induced Labeling of DNA (SMILing DNA). As a typical example we give a protocol for biotinylation of pBR322 plasmid DNA at the 5&#8217;-ATCGAT-3&#8217; sequence with the DNA MTase M.BseCI and the aziridine cofactor 6BAz in one step. Extension of the activated methyl group with unsaturated alkyl groups results in another class of AdoMet analogues which are used for methyltransferase-directed Transfer of Activated Groups (mTAG). Since the extended side chains are activated by the sulfonium center and the unsaturated bond, these cofactors are called double-activated AdoMet analogues. These analogues not only function as cofactors for DNA MTases, like the aziridine cofactors, but also for RNA, protein and small molecule MTases. They are typically used for enzymatic modification of MTase substrates with unique functional groups which are labeled with reporter groups in a second chemical step. This is exemplified in a protocol for fluorescence labeling of histone H3 protein. A small propargyl group is transferred from the cofactor analogue SeAdoYn to the protein by the histone H3 lysine 4 (H3K4) MTase Set7/9 followed by click labeling of the alkynylated histone H3 with TAMRA azide. MTase-mediated labeling with cofactor analogues is an enabling technology for many exciting applications including identification and functional study of MTase substrates as well as DNA genotyping and methylation detection.

DNA and proteins are sequence-specifically labeled with affinity or fluorescent reporter groups using DNA or protein methyltransferases and synthetic cofactor analogues. Depending on the cofactor specificity of the enzymes, aziridine or double activated cofactor analogues are employed for one- or two-step labeling.

Specifici Sequenza Etichettatura degli acidi nucleici e proteine ​​con metiltransferasi e cofattore Analoghi

S-Adenosyl-l-methionine (AdoMet or SAM)-dependent methyltransferases (MTase) catalyze the transfer of the activated methyl group from AdoMet to specific ...

Simple Method for Fluorescence DNA In Situ Hybridization to Squashed Chromosomes

DNA in situ hybridization (DNA ISH) is a commonly used method for mapping sequences to specific chromosome regions. This approach is particularly effective at mapping highly repetitive sequences to heterochromatic regions, where computational approaches face prohibitive challenges. Here we describe a streamlined protocol for DNA ISH that circumvents formamide washes that are standard steps in other DNA ISH protocols. Our protocol is optimized for hybridization with short single strand DNA probes that carry fluorescent dyes, which effectively mark repetitive DNA sequences within heterochromatic chromosomal regions across a number of different insect tissue types. However, applications may be extended to use with larger probes and visualization of single copy (non-repetitive) DNA sequences. We demonstrate this method by mapping several different repetitive sequences to squashed chromosomes from Drosophila melanogaster neural cells and Nasonia vitripennis spermatocytes. We show hybridization patterns for both small, commercially synthesized probes and for a larger probe for comparison. This procedure uses simple laboratory supplies and reagents, and is ideal for investigators who have little experience with performing DNA ISH.

Simple Method for Fluorescence DNA In Situ Hybridization to Squashed Chromosomes

Here, we present a simple method for performing fluorescence DNA in situ hybridization (DNA ISH) to visualize repetitive heterochromatic sequences on slide-mounted chromosomes. The method requires minimal reagents and it is versatile for use with short or long probes, different tissues, and detection with fluorescence or non-fluorescence-based signals.

Metodo semplice per DNA fluorescenza<em&gt; In Situ</em&gt; Ibridazione di cromosomi Squashed

DNA in situ hybridization (DNA ISH) is a commonly used method for mapping sequences to specific chromosome regions. This approach is particularly ...

Simple Bulk Readout of Digital Nucleic Acid Quantification Assays

Digital assays are powerful methods that enable detection of rare cells and counting of individual nucleic acid molecules. However, digital assays are still not routinely applied, due to the cost and specific equipment associated with commercially available methods. Here we present a simplified method for readout of digital droplet assays using a conventional real-time PCR instrument to measure bulk fluorescence of droplet-based digital assays.
We characterize the performance of the bulk readout assay using synthetic droplet mixtures and a droplet digital multiple displacement amplification (MDA) assay. Quantitative MDA particularly benefits from a digital reaction format, but our new method&#160;applies to any digital assay. For established digital assay protocols such as digital PCR, this method serves to speed up and simplify assay readout.
Our bulk readout methodology brings the advantages of partitioned assays without the need for specialized readout instrumentation. The principal limitations of the bulk readout methodology are reduced dynamic range compared with droplet-counting platforms and the need for a standard sample, although the requirements for this standard are less demanding than for a conventional real-time experiment. Quantitative whole genome amplification (WGA) is used to test for contaminants in WGA reactions and is the most sensitive way to detect the presence of DNA fragments with unknown sequences, giving the method great promise in diverse application areas including pharmaceutical quality control and astrobiology.

We describe an endpoint digital assay for quantifying nucleic acids with a simplified (analog) readout. We measure bulk fluorescence of droplet-based digital assays using a standard qPCR machine rather than specialized instrumentation and confirm our results by microscopy.

Semplice Lettura di massa di Digital acido nucleico quantificazione Assays

Digital assays are powerful methods that enable detection of rare cells and counting of individual nucleic acid molecules. However, digital assays are ...

Efficient Nucleic Acid Extraction and 16S rRNA Gene Sequencing for Bacterial Community Characterization

There is a growing appreciation for the role of microbial communities as critical modulators of human health and disease. High throughput sequencing technologies have allowed for the rapid and efficient characterization of bacterial communities using 16S rRNA gene sequencing from a variety of sources. Although readily available tools for 16S rRNA sequence analysis have standardized computational workflows, sample processing for DNA extraction remains a continued source of variability across studies. Here we describe an efficient, robust, and cost effective method for extracting nucleic acid from swabs. We also delineate downstream methods for 16S rRNA gene sequencing, including generation of sequencing libraries, data quality control, and sequence analysis. The workflow can accommodate multiple samples types, including stool and swabs collected from a variety of anatomical locations and host species. Additionally, recovered DNA and RNA can be separated and used for other applications, including whole genome sequencing or RNA-seq. The method described allows for a common processing approach for multiple sample types and accommodates downstream analysis of genomic, metagenomic and transcriptional information.

We describe an efficient, robust, and cost effective method for extracting nucleic acid from swabs for characterization of bacterial communities using 16S rRNA gene amplicon sequencing. The method allows for a common processing approach for multiple sample types and accommodates a number of downstream analytic processes.

Efficiente Nucleic Acid Estrazione e 16S rRNA sequenziamento del gene per batterica Comunità Caratterizzazione

There is a growing appreciation for the role of microbial communities as critical modulators of human health and disease. High throughput sequencing ...

A Rapid, Scalable Method for the Isolation, Functional Study, and Analysis of Cell-derived Extracellular Matrix

The extracellular matrix (ECM) is recognized as a diverse, dynamic, and complex environment that is involved in multiple cell-physiological and pathological processes. However, the isolation of ECM, from tissues or cell culture, is complicated by the insoluble and cross-linked nature of the assembled ECM and by the potential contamination of ECM extracts with cell surface and intracellular proteins. Here, we describe a method for use with cultured cells that is rapid and reliably removes cells to isolate a cell-derived ECM for downstream experimentation.
Through use of this method, the isolated ECM and its components can be visualized by in situ immunofluorescence microscopy. The dynamics of specific ECM proteins can be tracked by tracing the deposition of a tagged protein using fluorescence microscopy, both before and after the removal of cells. Alternatively, the isolated ECM can be extracted for biochemical analysis, such as sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting. At larger scales, a full proteomics analysis of the isolated ECM by mass spectrometry can be conducted. By conducting ECM isolation under sterile conditions, sterile ECM layers can be obtained for functional or phenotypic studies with any cell of interest. The method can be applied to any adherent cell type, is relatively easy to perform, and can be linked to a wide repertoire of experimental designs.

The extracellular matrix plays a major role in defining the microenvironment of cells and in modulating cell behavior and phenotype. We describe a rapid method for the isolation of cell-derived extracellular matrix, which can be adapted to different scales for microscopic, biochemical, proteomic, or functional studies.

L'isolamento di filtrazione di acidi nucleici: DNA metodo di estrazione semplice e veloce

Riepilogo

Esplora Altri Video

Capitoli in questo video

NanoDrop microvolumi quantificazione degli acidi nucleici

Determinazione concentrazione di acidi nucleici e proteine Utilizzando il Micro-volume Bio-spec Spettrofotometro Nano

Estrazione DNA da materiale in paraffina per le analisi genetiche ed epigenetiche

Elettricità-Free, Nucleic Acid sequenziale e isolamento delle proteine

Isolamento rapida e purificazione di mitocondri per il trapianto da tessuto Dissociazione e Filtrazione Differenziale

Specifici Sequenza Etichettatura degli acidi nucleici e proteine con metiltransferasi e cofattore Analoghi

Metodo semplice per DNA fluorescenza

Semplice Lettura di massa di Digital acido nucleico quantificazione Assays

Efficiente Nucleic Acid Estrazione e 16S rRNA sequenziamento del gene per batterica Comunità Caratterizzazione

A Rapid, metodo scalabile per l'isolamento, studio funzionale, e analisi di Cell-derivati matrice extracellulare

L'isolamento di filtrazione di acidi nucleici: DNA metodo di estrazione semplice e veloce

Riepilogo

Esplora Altri Video

Capitoli in questo video

NanoDrop microvolumi quantificazione degli acidi nucleici

Determinazione concentrazione di acidi nucleici e proteine ​​Utilizzando il Micro-volume Bio-spec Spettrofotometro Nano

Estrazione DNA da materiale in paraffina per le analisi genetiche ed epigenetiche

Elettricità-Free, Nucleic Acid sequenziale e isolamento delle proteine

Isolamento rapida e purificazione di mitocondri per il trapianto da tessuto Dissociazione e Filtrazione Differenziale

Specifici Sequenza Etichettatura degli acidi nucleici e proteine ​​con metiltransferasi e cofattore Analoghi

Metodo semplice per DNA fluorescenza

Semplice Lettura di massa di Digital acido nucleico quantificazione Assays

Efficiente Nucleic Acid Estrazione e 16S rRNA sequenziamento del gene per batterica Comunità Caratterizzazione

A Rapid, metodo scalabile per l'isolamento, studio funzionale, e analisi di Cell-derivati ​​matrice extracellulare

Determinazione concentrazione di acidi nucleici e proteine Utilizzando il Micro-volume Bio-spec Spettrofotometro Nano

Specifici Sequenza Etichettatura degli acidi nucleici e proteine con metiltransferasi e cofattore Analoghi

A Rapid, metodo scalabile per l'isolamento, studio funzionale, e analisi di Cell-derivati matrice extracellulare