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.

Ethics statement: The whole blood specimens used in this study are not considered to be research involving human subjects. The specimens were obtained for clinical diagnostic purposes, which were satisfied, and the remaining portion of these specimens was provided for the FINA research assay. The specimens were coded such that the investigators were not able to readily ascertain the identity of individuals.
1. Preparation of FINA Sample Preparation Module
<ol>
	<li>Prepare blotting pad by cutting a 35 x 35 mm<su...

<ol>
	<li>Byrnes, S. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=One-step+purification+and+concentration+of+DNA+in+porous+membranes+for+point-of-care+applications.">One-step purification and concentration of DNA in porous membranes for point-of-care applications.</a> Lab Chip. 15 (12), 2647-2659 (2015).</li><li>Connelly, J. T., Rolland, J. P., Whitesides, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term="Paper+Machine"+for+Molecular+Diagnostics.">"Paper Machine" for Molecular Diagnostics.</a> Anal Chem. 87 (15), 7595-7601 (2015).</li><li>Govindarajan, A. V., Ramachandran, S., Vigil, G. D., Yager, P., Bohringer, K. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+low+cost+point-of-care+viscous+sample+preparation+device+for+molecular+diagnosis+in+the+developing+world;+an+example+of+microfluidic+origami.">A low cost point-of-care viscous sample preparation device for molecular diagnosis in the developing world; an example of microfluidic origami.</a> Lab Chip. 12 (1), 174-181 (2012).</li><li>Linnes, J. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Paper-based+molecular+diagnostic+for.">Paper-based molecular diagnostic for.</a> RSC Adv. 4 (80), 42245-42251 (2014).</li><li>Rodriguez, N. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Paper-Based+RNA+Extraction,+in+Situ+Isothermal+Amplification,+and+Lateral+Flow+Detection+for+Low-Cost,+Rapid+Diagnosis+of+Influenza+A+(H1N1)+from+Clinical+Specimens.">Paper-Based RNA Extraction, in Situ Isothermal Amplification, and Lateral Flow Detection for Low-Cost, Rapid Diagnosis of Influenza A (H1N1) from Clinical Specimens.</a> Anal Chem. 87 (15), 7872-7879 (2015).</li><li>Peeling, R. W., Holmes, K. K., Mabey, D., Ronald, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+tests+for+sexually+transmitted+infections+(STIs):+the+way+forward.">Rapid tests for sexually transmitted infections (STIs): the way forward.</a> Sex Transm Infect. 82, 1-6 (2006).</li><li>Mabey, D., Peeling, R. W., Ustianowski, A., Perkins, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diagnostics+for+the+developing+world.">Diagnostics for the developing world.</a> Nat Rev Microbiol. 2 (3), 231-240 (2004).</li><li>Jangam, S. R., Agarwal, A. K., Sur, K., Kelso, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+point-of-care+PCR+test+for+HIV-1+detection+in+resource-limited+settings.">A point-of-care PCR test for HIV-1 detection in resource-limited settings.</a> Biosens Bioelectron. 42, 69-75 (2013).</li><li>Jangam, S. R., Yamada, D. H., McFall, S. M., Kelso, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+point-of-care+extraction+of+human+immunodeficiency+virus+type+1+proviral+DNA+from+whole+blood+for+detection+by+real-time+PCR.">Rapid point-of-care extraction of human immunodeficiency virus type 1 proviral DNA from whole blood for detection by real-time PCR.</a> J Clin Microbiol. 47 (8), 2363-2368 (2009).</li><li>Kelso, D. M., Jangam, S., Yamada, D., McFall, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Google Patents. , (2012).</li><li>McFall, S. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Simple+and+Rapid+DNA+Extraction+Method+from+Whole+Blood+for+Highly+Sensitive+Detection+and+Quantitation+of+HIV-1+Proviral+DNA+by+Real-Time+PCR.">A Simple and Rapid DNA Extraction Method from Whole Blood for Highly Sensitive Detection and Quantitation of HIV-1 Proviral DNA by Real-Time PCR.</a> Journal of Virological Methods. 214, 37-42 (2015).</li><li>Boom, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+and+simple+method+for+purification+of+nucleic+acids.">Rapid and simple method for purification of nucleic acids.</a> J Clin Microbiol. 28 (3), 495-503 (1990).</li><li>Radstrom, P., Knutsson, R., Wolffs, P., Lovenklev, M., Lofstrom, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pre-PCR+processing:+strategies+to+generate+PCR-compatible+samples.">Pre-PCR processing: strategies to generate PCR-compatible samples.</a> Mol Biotechnol. 26 (2), 133-146 (2004).</li><li>Schrader, C., Schielke, A., Ellerbroek, L., Johne, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PCR+inhibitors+-+occurrence,+properties+and+removal.">PCR inhibitors - occurrence, properties and removal.</a> J Appl Microbiol. 113 (5), 1014-1026 (2012).</li><li>Folks, T. M., Powell, D. M., Martin, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Google Patents. , (1991).</li><li>Violari, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+Antiretroviral+Therapy+and+Mortality+among+HIV-Infected+Infants.">Early Antiretroviral Therapy and Mortality among HIV-Infected Infants.</a> N Engl J Med. 359 (21), 2233-2244 (2008).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> World Health Organization. , (2010).</li><li>Gruner, N., Stambouli, O., Ross, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dried+blood+spots--preparing+and+processing+for+use+in+immunoassays+and+in+molecular+techniques.">Dried blood spots--preparing and processing for use in immunoassays and in molecular techniques.</a> J Vis Exp. (97), (2015).</li><li>Maiers, T. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+investigation+of+fingerstick+blood+collection+for+point-of-care+HIV-1+viral+load+monitoring+in+South+Africa.">An investigation of fingerstick blood collection for point-of-care HIV-1 viral load monitoring in South Africa.</a> S Afr Med J. 105 (3), 228-231 (2015).</li></ol>

EID linked to rapid treatment access has been demonstrated to reduce infant mortality due to HIV infection 16. Because of the persistence of maternal antibodies in an infant&#39;s blood, rapid HIV antibody tests have limited utility in determining the status of HIV-exposed infants. The WHO recommends that all infants born to HIV-1 positive mothers should be tested at 4-6 weeks of age, using a virological test 17. We have reported the development of an assay for the detection and quantitation of HIV-...

Several reports have discussed the development of paper- or membrane-based extraction methods for use in point-of-care (POC) devices 1-5 with the aim of making the exquisite sensitivity and specificity of molecular diagnostics available to all. The World Health Organization (WHO) Sexually Transmitted Diseases Diagnostics Initiative coined the term ASSURED (Affordable, Sensitive, Specific, User-friendly, Rapid and Robust, Equipment-free and Delivered to those who need it) to describe the ideal characteristics of a POC test 6. Of these guidelines, the equipment-free characteristic is particularly challenging to achieve for molecular diagnostics. However, every innovation in the field will advance the goal of reaching those in most need, and there is hope for near-term improvements in test performance by adapting existing technology 7.
Here we describe a simple protocol for extracting DNA from whole blood that does not require complex chemistry or laboratory instrumentation. The FINA (filtration isolation of nucleic acids) sample preparation method was originally developed to extract leukocyte DNA from whole blood in order to detect the HIV-1 provirus as part of a sample-to-answer point-of-care (POC) quantitative PCR (qPCR) early infant diagnostic (EID) platform for use in limited resource settings 8-11. FINA extraction differs from conventional purification methods which use silica membranes or silica-coated paramagnetic particles to reversibly bind DNA in the presence of chaotropic agents 12. Instead, FINA uses vertical filtration via a separation membrane to extract cellular DNA from whole blood directly. The membrane containing the entrapped DNA can be placed directly in a PCR tube and either immediately used in a PCR reaction or air dried for later amplification 9. No chaotropic agents, phenol, or alcohols are used in the sample extraction, eliminating the extensive washing steps needed to remove potent qPCR inhibitors derived from the extraction process 13,14.
The FINA membrane can capture either cells 9 or genomic DNA liberated by cell lysis 11 before the specimen is added to the membrane. For the cell capture, whole blood is added directly to the membrane. The cells are subsequently lysed in the membrane by adding 10 mM NaOH. The advantage to this method is that it involves only 3 steps: 1) sample addition; 2) cell lysis/wash and 3) filter disk placement in qPCR tube. The drawback to this method is that the membrane can only hold a defined number of cells directly proportional to the diameter of the membrane disk. To reach the limit of detection required for EID, 100 &#181;l of whole blood is required for sample input which entails a filter that is too large to be placed in a qPCR tube. Lysing the blood cells with detergent before adding the sample to the collection membrane adds a processing step, but allows the use of a smaller filter for the same sample size. We were able to demonstrate high reproducibility, single copy detection, and quantification of as little as 10 copies of HIV-1 proviral DNA from 100 &#181;l of blood using this test configuration 11.
In this report, we describe the FINA protocol as originally developed for laboratory use. The membrane/filter sandwich known as the FINA sample preparation module can be prepared in large batches and stockpiled for later use. When specimens are to be extracted this process takes 2 min and can be performed in varying size batches. The qPCR can be run immediately or the filters containing the embedded DNA can be stored until it is convenient to perform qPCR. This method is very convenient for routine analysis of specimens in both low and high resource settings.

<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, filtration isolation of nucleic acids, is a novel extraction method which utilizes vertical filtration via a separation membrane and absorbent pad to extract cellular DNA from whole blood in less than 2 min. The blood specimen is treated with detergent, mixed briefly and applied by pipet to the separation membrane. The lysate wicks into the blotting pad due to capillary action, capturing the genomic DNA on the surface of the separation membrane. The extracted DNA is retained on the membrane during a simple wash step wherein PCR inhibitors are wicked into the absorbent blotting pad. The membrane containing the entrapped DNA is then added to the PCR reaction without further purification. This simple method does not require laboratory equipment and can be easily implemented with inexpensive laboratory supplies. Here we describe a protocol for highly sensitive detection and quantitation of HIV-1 proviral DNA from 100 &#181;l whole blood as a model for early infant diagnosis of HIV that could readily be adapted to other genetic targets.

The workflow for extracting proviral DNA from whole blood spiked with 8e5-LAV cells is shown in Figure 1. Figure 2 shows the FINA sample module and prepared qPCR tube. This method allows for efficient amplification of HIV-1 provirus from 8e5-LAV cells at different copy numbers, as shown in the standard curve of contrived specimens (Figure 3). PCR had an efficiency of 103%, as calculated from Efficiency = -1+10(-1/slope). Equati...

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

We describe here a simple and rapid paper-based DNA extraction method of HIV proviral DNA from whole blood detected by quantitative PCR. This protocol can be extended for use in detecting other genetic markers or using alternative amplification methods.

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

The overall goal of this FINA method which stands for filtration isolation of nucleic acids is to isolate genomic DNA for PCR amplification from complex specimens quickly and simply without the use of laboratory instrumentation such as a centrifuge. This rapid paper based DNA extraction method for HIV pro viral DNA was developed for incorporation into a quantitative PCR diagnostic device. The main advantage of this technique is that the sample prep modules and PCR tubes can be prepared in advance so the DNA extraction takes less than two minutes.We first developed this method to be incorporated into a sample to answer early infant diagnostic test for HIV-1 from heelstick blood. Generally individuals new to this method may struggle because removing the backing of the double sided tape that holds the cell capture membrane to the side of the PCR tube requires practice. Demonstrating the procedure will be Jen Reed, a senior technician from my lab.Begin this procedure by preparing the materials needed for assembling the FINA sample preparation module. Prepare the blotting pad by cutting a 35 by 35 millimeter square of a 2.6 millimeter thick 100%cotton pad. Prepare plastic paraffin film with a paper backing tape.Cut a 25 by 50 mm piece of paraffin film and then use a hammer to punch a 7.14 mm diameter hole in the center of the tape. Prepare a bound glass blood separator membrane or capture disk by using a hammer to punch an 8.35 mm diameter circle. To assemble the FINA sample preparation module use forceps to place the capture disc in the center of the blotting pad.Remove the backing of the paraffin film tape and place it over the capture disc so that the whole of the paraffin tape leaves the center of the capture disc exposed. Ensure maximum contact between the disc and the blotting pad by stretching the paraffin tape slightly to cover the blotting pad and pressing the paraffin tape firmly to stick it to the blotting pad around all edges of the disc. A 5.1 mm tape disc is required to anchor the capture membrane to the side of qPCR tube to prevent the membrane from blocking the fluorescence detection of the real time PCR instrument.Prepare this tape disc by using a hammer to punch a circle of double coated polyester diagnostic tape from a sheet of the tape. Using forceps remove one side of the tape sticker liner. Place the tape into a 200 microliter PCR tube, sticking it onto one side and toward the bottom of the tube.Remove the second sticker liner. The tube is now ready to receive the prepared disc. The detection of proviral DNA from whole blood spiked with 8e5 LAV cells will be demonstrated in this video.Thaw out the previously frozen 8e5 LAV cell pellets on ice. Prepare a serial dilution of the cells in freezing medium with concentrations ranging from 1 to 400 cells per microliter. Pipette 10 microliters of cells from each of the dilutions into a micro centrifuge tube.Add 100 microliters of fresh HIV-1 negative eDTA treated whole blood sample to each tube to create a standard curve. Mix by gently flicking the bottom of the tube five times. To begin the DNA extraction procedure, lyse the blood cells by adding triton X-100 to a final concentration of one percent.Mix by gently flicking the bottom of the tube five times. The blood should turn a translucent red. Pipette the entire blood specimen onto the capture disc.A water tight seal between the paraffin tape and the capture membrane ensures that the blood flows through the membrane. And good contact between the contact membrane and blotting pad assembly ensures rapid sample wicking. Allow the sample to soak through the filter.The blood lysate has soaked into the capture disc and the disc appears mat. Add 600 to 1, 000 microliters of 10 mmol sodium hydroxide dropwise onto the capture disc. The filter will change from red to white, indicating clearance of hemoglobin.Next separate the disc from the blotting pad and apply the disc to the tape in the prepared PCR tube. After the filter is placed in the PCR tube analyze the sample right away. Prepare the qPCR master mix as described in the protocol text and be sure that the filter is completely covered with the master mix and stays fixed to the side of the tube throughout the reaction.Perform amplification using a real time PCR device as described in the protocol text. Alternatively if qPCR cannot be performed immediately dry the filter overnight in a box containing calcium sulfate dessicant. On the following day, cap and place the PCR tube in a foiled pouch with silica dessicant and store at room temperature until ready for analysis.This method allows for efficient amplification of HIV-1 provirus from 8e5 LAV cells at different copy numbers. The solid lines are amplification plots obtained from four replicates of HIV-1 negative whole blood spiked with 4, 000, 400, 40 and 10 8e5 LAV cells with 500 copies per reaction of the internal control. The dotted line indicates the threshold.This next graph shows standard curves of quantitation cycle values calculated from the amplification plot vs. log copy number of 8e5 LAV cells per 100 microliters of whole blood. The calculated PCR efficiency was 103%The HIV proviral DNA standard curve replicates also have highly reproducible amplification.Additionally the presence of an internal control of 500 copies of hydroxy pyruvate reductase shows that there is no PCR inhibition resulting from extracting blood with this method independent of the number of copies of HIV-1 provirus present. The solid lines represent the amplification plots and the dotted line represents the threshold. Following this procedure PCR targets can be amplified from various specimen types to detect your gene of interest.For example cheek swabs for pharmacogenetic screening or blood for rapid genotyping of animal models. Don't forget that working with blood borne pathogens can be extremely hazardous and universal precautions should always be taken while performing this procedure.

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Research

JoVE Journal

Biology

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.

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

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.

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

qPCR Is a Sensitive and Rapid Method for Detection of Cytomegaloviral DNA in Formalin-fixed, Paraffin-embedded Biopsy Tissue

It is crucial to identify cytomegalovirus (CMV) infection in the gastrointestinal (GI) tract of immunosuppressed patients, given their greater risk for developing severe infection. Many laboratory methods for the detection of CMV infection have been developed, including serology, viral culture, and molecular methods. Often, these methods reflect systemic involvement with CMV and do not specifically identify local tissue involvement. Therefore, detection of CMV infection in the GI tract is frequently done by traditional histology of biopsy tissue. Hematoxylin and eosin (H&#38;E) staining in conjunction with immunohistochemistry (IHC) have remained the mainstays of examining these biopsies. H&#38;E and IHC sometimes result in atypical (equivocal) staining patterns, making interpretation difficult. It was shown that quantitative polymerase chain reaction (qPCR) for CMV can successfully be performed on formalin-fixed, paraffin-embedded (FFPE) biopsy tissue for very high sensitivity and specificity. The goal of this protocol is to demonstrate how to perform qPCR testing for the detection of CMV in FFPE biopsy tissue in a clinical laboratory setting. This method is likely to be of great benefit for patients in cases of equivocal staining for CMV in GI biopsies.

This protocol describes qPCR detection of cytomegalovirus in formalin-fixed, paraffin-embedded biopsy tissue, which is rapid, sensitive, specific, and useful for interpreting equivocal hematoxylin and eosin or immunohistochemical staining patterns.

It is crucial to identify cytomegalovirus (CMV) infection in the gastrointestinal (GI) tract of immunosuppressed patients, given their greater risk for ...

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.

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.

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

A Hybrid DNA Extraction Method for the Qualitative and Quantitative Assessment of Bacterial Communities from Poultry Production Samples

The efficacy of DNA extraction protocols can be highly dependent upon both the type of sample being investigated and the types of downstream analyses performed. Considering that the use of new bacterial community analysis techniques (e.g., microbiomics, metagenomics) is becoming more prevalent in the agricultural and environmental sciences and many environmental samples within these disciplines can be physiochemically and microbiologically unique (e.g., fecal and litter/bedding samples from the poultry production spectrum), appropriate and effective DNA extraction methods need to be carefully chosen. Therefore, a novel semi-automated hybrid DNA extraction method was developed specifically for use with environmental poultry production samples. This method is a combination of the two major types of DNA extraction: mechanical and enzymatic. A two-step intense mechanical homogenization step (using bead-beating specifically formulated for environmental samples) was added to the beginning of the &#8220;gold standard&#8221; enzymatic DNA extraction method for fecal samples to enhance the removal of bacteria and DNA from the sample matrix and improve the recovery of Gram-positive bacterial community members. Once the enzymatic extraction portion of the hybrid method was initiated, the remaining purification process was automated using a robotic workstation to increase sample throughput and decrease sample processing error. In comparison to the strict mechanical and enzymatic DNA extraction methods, this novel hybrid method provided the best overall combined performance when considering quantitative (using 16S rRNA qPCR) and qualitative (using microbiomics) estimates of the total bacterial communities when processing poultry feces and litter samples.

A novel semi-automated hybrid DNA extraction method for use with environmental poultry production samples was developed and demonstrated improvements over a common mechanical and enzymatic extraction method in terms of the quantitative and qualitative estimates of the total bacterial communities.

The efficacy of DNA extraction protocols can be highly dependent upon both the type of sample being investigated and the types of downstream analyses ...

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.

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.

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.

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.