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 mm2 of a 2.6 mm thick 100% cotton pad. Cut one pad per specimen.</li>
	<li>Prepare plastic paraffin film with a paper backing tape by a cutting piece of paraffin film (25 mm (h) by 50 mm (w)) and then punching a 7.14 mm diameter hole in the center of the tape using a hammer driven hole punch. Prepare one tape per specimen.</li>
	<li>Prepare a bound glass blood separator membrane (capture membrane) disk by punching a 8.35 mm diameter circle using a hammer driven hole punch. Punch one disk per specimen. The disk has a particle retention capability of 2.3 &#181;m.</li>
	<li>Assemble the FINA sample preparation module by placing the capture disk in the center of the blotting pad using forceps. Place the paraffin film tape over the capture disk so that the hole of the paraffin tape leaves the center of the capture disk exposed.</li>
	<li>Ensure maximum contact between the disk 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 disk.</li>
	<li>Make as many modules as needed for the experiment or stockpile for future use.</li>
</ol>
2. Preparation of qPCR Tube
<ol>
	<li>Prepare a 5.1 mm tape disk that will anchor the cell capture membrane to the side of the qPCR tube to prevent the membrane from blocking the fluorescence detection by punching a circle of double-coated polyester diagnostic tape with a hammer driven punch from a sheet of the tape. Prepare one tape disk per specimen.</li>
	<li>Remove one side of the tape&#39;s sticker liner. Place the tape into a 200 &#181;l 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 prepared disk.</li>
	<li>Produce as many tubes as needed for the experiment or stockpile for future use.</li>
</ol>
3. Contrive Blood Specimen
Note: If a genuine test specimen is being prepared, proceed to step 4.
<ol>
	<li>Thaw 8e5-LAV cells 15 harboring a single copy of the HIV-1 provirus obtained from the Virology Quality Assurance Laboratory (VQA; Rush Presbyterian/St. Luke&#39;s Medical Center, Chicago, IL). 
	Note: They are stored as frozen cell pellets at a concentration of 4,000 cells/&#181;l. Cell count was verified as described previously 9.</li>
	<li>Prepare serial dilution in freezing medium (90% fetal bovine serum, 10% dimethyl sulfoxide) to concentrations ranging from 1 to 400 cells/&#181;l.</li>
	<li>Pipet 10 &#181;l cells from each of the serial dilutions into a microcentrifuge tube. Add 100 &#181;l of fresh HIV-1 negative EDTA treated whole blood sample to each tube to create a standard curve of 8e5-LAV copy numbers 10-4,000. Mix by gently flicking the bottom of the tube 5 times.</li>
</ol>
4. Perform FINA Extraction
<ol>
	<li>Lyse blood cells by adding Triton-X100) to a final concentration of 1% (11 &#181;l 10% Triton-X100 to 110 &#181;l whole blood and cells from step 3.3).</li>
	<li>Mix by gently flicking the bottom of the tube 5 times. The blood should turn translucent red.</li>
	<li>Pipet all of the blood specimen onto the capture disk. 
	Note: 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 capture membrane and blotting pad assembly ensures rapid sample wicking.</li>
	<li>Allow sample to soak through the filter. 
	Note: The blood lysate wicks into the blotting pad due to capillary action, capturing the genomic DNA on the surface of the capture disk. The lysate has soaked into the disk when it appears matte.</li>
	<li>Add 600-1,000 &#181;l 10 mM NaOH drop-wise onto the capture disk. 
	Note: The wash buffer can also be added as a bulk addition of 600 &#181;l. The buffer will form a droplet on the hydrophobic paraffin tape and wick through the hole to the disk in approximately 20 sec. The filter will change from red to white indicating clearance of hemoglobin.</li>
	<li>Separate the disk from the blotting pad with forceps and apply the disk to the tape in the prepared PCR tube. If there is any red color left on the filter add 50-100 &#181;l of wash buffer to clear the filter before placing in the tube. 
	Note: Infrequently, excess pressure applied during preparation of the FINA modules results in partitioning of the membrane layers during separation from the blotting pad. Discard these disks and prepare a fresh sample.</li>
	<li>After the filter is placed in the PCR tube, analyze the samples right away. Alternatively, dry overnight in a box containing calcium sulfate desiccant, then cap and place in a foil pouch with silica desiccant and store until ready for analysis.</li>
</ol>
5. qPCR Reaction
<ol>
	<li>Prepare 100 &#181;l qPCR master mix per reaction using HIV specific primers and probes as described previously 9,11. Include an internal amplification control to monitor for the presence of amplification inhibitors and to verify that a negative sample is a true negative. Be sure that filter is completely covered with master mix and that the filter stays fixed to side of tube throughout the reaction.</li>
	<li>Perform amplification using a real-time PCR device as previously described 9.</li>
</ol>

<ol><li>Byrnes, S. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((One-step+purification+and+concentration+of+DNA+in+porous+membranes+for+point-of-care+applications%5BTitle%5D)+AND+%22Lab+Chip%22%5BJournal%5D)">One-step purification and concentration of DNA in porous membranes for point-of-care applications.</a> Lab Chip. 15 (12), 2647-2659 (2015).</li>
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<li>Peeling, R. W., Holmes, K. K., Mabey, D., Ronald, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Rapid+tests+for+sexually+transmitted+infections+%28STIs%29%3A+the+way+forward%5BTitle%5D)+AND+%22Sex+Transm+Infect%22%5BJournal%5D)">Rapid tests for sexually transmitted infections (STIs): the way forward.</a> Sex Transm Infect. 82, Suppl 5 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/pubmed?term=((Diagnostics+for+the+developing+world%5BTitle%5D)+AND+%22Nat+Rev+Microbiol%22%5BJournal%5D)">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/pubmed?term=((A+point-of-care+PCR+test+for+HIV-1+detection+in+resource-limited+settings%5BTitle%5D)+AND+%22Biosens+Bioelectron%22%5BJournal%5D)">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/pubmed?term=((Rapid+point-of-care+extraction+of+human+immunodeficiency+virus+type+1+proviral+DNA+from+whole+blood+for+detection+by+real-time+PCR%5BTitle%5D)+AND+%22J+Clin+Microbiol%22%5BJournal%5D)">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. Google Patents. , (2012).</li>
<li>McFall, S. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?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%5BTitle%5D)+AND+%22Journal+of+Virological+Methods%22%5BJournal%5D)">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/pubmed?term=((Rapid+and+simple+method+for+purification+of+nucleic+acids%5BTitle%5D)+AND+%22J+Clin+Microbiol%22%5BJournal%5D)">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/pubmed?term=((Pre-PCR+processing%3A+strategies+to+generate+PCR-compatible+samples%5BTitle%5D)+AND+%22Mol+Biotechnol%22%5BJournal%5D)">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/pubmed?term=((PCR+inhibitors+-+occurrence%2C+properties+and+removal%5BTitle%5D)+AND+%22J+Appl+Microbiol%22%5BJournal%5D)">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. Google Patents. , (1991).</li>
<li>Violari, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Early+Antiretroviral+Therapy+and+Mortality+among+HIV-Infected+Infants%5BTitle%5D)+AND+%22N+Engl+J+Med%22%5BJournal%5D)">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://scholar.google.com/scholar?hl=en&safe=off&q=%22World+Health+Organization%22">World Health Organization</a>. , World Health Organization. Geneva, Switzerland. (2010).</li>
<li>Gruner, N., Stambouli, O., Ross, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Dried+blood+spots--preparing+and+processing+for+use+in+immunoassays+and+in+molecular+techniques%5BTitle%5D)+AND+%22J+Vis+Exp%22%5BJournal%5D)">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/pubmed?term=((An+investigation+of+fingerstick+blood+collection+for+point-of-care+HIV-1+viral+load+monitoring+in+South+Africa%5BTitle%5D)+AND+%22S+Afr+Med+J%22%5BJournal%5D)">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>

The authors have nothing to disclose.

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-1 proviral DNA in whole blood specimens that is capable of single copy detection with demonstrated 100% sensitivity and specificity in a small field evaluation of 61 South African infants 11. One hundred microliters or more of whole blood can be collected via finger or heel stick and processed via the FINA modules 18,19. These blood samples can then be stored for at least one month before amplification making this method ideal for testing infants that are far from laboratories.
In the study presented in Figure 3, a standard curve of contrived specimens from HIV-1 negative whole blood spiked with thawed cells harboring HIV-1 proviral DNA is depicted. A caveat to this study is that some of the cells added to the sample could have already lysed due to the freeze/thaw, which could have potentially improved the detection level of the target. A more stringent experimental design would have been to have contrived the specimen using freshly cultured cells which would have more accurately mimicked whole blood.
The FINA sample prep method was designed for incorporation into an EID POC qPCR device (under development) for use in limited resource settings 8; however, the manual format described here can also be a useful addition to a laboratory&#39;s sample prep portfolio. FINA does not use the bind, wash and elute strategy used in dried blood spots and other paper-based extraction systems that dilutes the extracted nucleic acids, possibly leading to sub-optimal detection sensitivity 5. The FINA system is highly flexible and can be readily adapted for other genetic tests besides proviral HIV DNA detection. Lower volumes of blood or other biological specimens can be processed for abundant targets and higher volumes for less abundant ones. In our original embodiment of the FINA system, we capture the cells from whole blood using a blood separation membrane and then lyse them by the addition of 10 mM NaOH 9. This version has the advantage of one fewer user steps, but the cell number/blood volume that can be processed is smaller. In addition to the bound glass membrane used in this report, other blood separation or blood collection filter papers have been used successfully with this method, and several different quantitative PCR instruments have also been shown to amplify template embedded in the filter disk 9. The only stipulation is that the filter does not block the fluorescence reading of the real-time PCR instrument.
A significant limitation of FINA extraction is that there is a size constraint on the diameter of the capture membrane. A disk with a membrane larger than 9 mm cannot be placed in a 200 &#181;l qPCR tube without the membrane overlapping in the tube which limits the surface area exposed to the reaction mix. Additionally, the larger the disk diameter, the more qPCR reaction volume is required to cover the filter which increases the cost and turn-around-time of the assay. If the filter is too small to efficiently capture the DNA in the specimen, it may clog preventing efficient washing.
Using the bound glass capture membrane, we were able to capture only HIV-1 proviral DNA and not viral RNA from whole blood samples (data not shown). We do not know if alternative membranes or buffers could be used to promote isolation of RNA instead of or in addition to DNA. If the reader would like to only isolate DNA from the specimen this protocol does not require the enzymatic removal of RNA required with some protocols. Detecting both HIV-1 RNA and DNA in clinical samples would improve the sensitivity of EID and this lack of RNA detection is a limitation of our diagnostic test.
The most critical step in adapting the FINA process to a new assay is to validate the size of the filter to accommodate the amount of DNA, i.e., the number of cells, in the specimen to be tested. In general FINA extraction works best with specimens with smaller numbers of cells so that smaller capture disks can be used. To determine the binding capacity of the filter, simply add a second filter disk to the FINA module stacked below the collection membrane. Add the sample and wash the membranes as in steps 4.1-4.5. Place each of the DNA capture disks into separate qPCR tubes and amplify. Using a standard curve, determine the percent of input DNA captured on each filter. The size of the filter can be optimized to the needs of the assay. For the FINA proviral DNA assay described here, more than 99% of human genomic DNA is captured when cell lysate is added to the filter (unpublished observations) enabling superior detection capabilities.
In addition to EID, future applications of FINA could include the detection and quantitation of proviral DNA as a biomarker for HIV-1 disease monitoring with patients who have achieved viral suppression due to antiretroviral therapy. Treatment monitoring aimed at HIV eradication could be performed by screening other inputs besides peripheral blood for reservoirs of HIV-1 infected cells. HIV proviral testing could also be used to screen vaccine trial subjects for true infection. The blood samples are stable during storage on FINA modules and can be collected under field conditions for later shipment to central laboratories for PCR analysis making this method ideal for epidemiological studies. In addition to HIV-1 detection, this highly flexible method could also be used to amplify precision medicine targets found in biological specimens such as pharmacogenetics screening or other SNP detection or for rapid genotyping of animal models. Isothermal amplification methods such as helicase dependent amplification (HDA) or loop-mediated isothermal amplification (LAMP) could also be used with FINA extracted template or the DNA-embedded on the filter could be used as template for conventional PCR amplification.

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><tbody><tr><td>Fusion 5</td><td>GE Healthcare Life Sciences</td><td>8151-9915</td><td></td></tr><tr><td>707 blotting pad</td><td>VWR International</td><td>28298-014</td><td></td></tr><tr><td>PARAFILM M</td><td>VWR International</td><td>52858-000</td><td></td></tr><tr><td>3M Double-Coated Polyester Diagnostic Tape&amp;nbsp;</td><td>3M Medical Specialties</td><td>9965</td><td></td></tr><tr><td>200 &amp;micro;l qPCR strip tubes</td><td>Agilent</td><td>401428</td><td></td></tr><tr><td>optical strip caps</td><td>Agilent</td><td>401425</td><td></td></tr><tr><td>Mx3005p qPCR System</td><td>Agilent</td><td>401456</td><td></td></tr><tr><td>sodium hydroxide</td><td>Sigma-Aldrich</td><td>221465</td><td>A.C.S. Reagent</td></tr><tr><td>Triton X-100</td><td>Sigma-Aldrich</td><td>T9284</td><td>BioXtra</td></tr><tr><td>dimethyl sulfoxide</td><td>Sigma-Aldrich</td><td>D8418</td><td>for molecular biology</td></tr><tr><td>fetal bovine serum, certified, U.S. origin</td><td>Thermo Fisher Scientific</td><td>16000-044</td><td></td></tr><tr><td>Hammer driven small hole punch 3/16&quot; hole diameter (5.1 mm)</td><td>McMaster Carr</td><td>3424A16</td><td></td></tr><tr><td>Hammer driven small hole punch 1/4&quot; hole diameter (7.14 mm)</td><td>McMaster Carr</td><td>3424A19</td><td></td></tr><tr><td>Hammer driven small hole punch 5/16&quot; 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). Equation of the line was y= -3.25x + 27.95, with a correlation of R&#178; = 0.996. The HIV proviral DNA standard curve replicates also have highly reproducible amplification, as evidenced in Table 1. Additionally, an internal control of 500 copies Hydroxypyruvate Reductase is present to show that there is no PCR inhibition resulting from extracting blood with FINA, independent of the number of copies of HIV-1 provirus present (Figure 4). IC amplification was efficiently obtained in all 18 samples tested, with an average Cq of 21.92 &#177; 0.25 and a CV of 1%.
<img alt="Figure 1" src="/files/ftp_upload/54289/54289fig1.jpg" /> 
Figure 1: Workflow for detecting proviral DNA from whole blood spiked with 8e5-LAV cells to qPCR. (A) From left to right, the prepared FINA module, after blood is added to the capture membrane and after wash buffer was added. (B) qPCR tubes with capture disks stuck to double-coated tape with qPCR master mix added. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54289/54289fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/54289/54289fig2.jpg" /> 
Figure 2: FINA in-house module and qPCR tube preparation. (A) An 8.35-mm diameter capture membrane disk was sandwiched between a square 707 blotting pad and a thin sheet of paraffin tape containing a 7.14-mm-diameter hole in the center such that the hole of the paraffin tape was centered on the capture disk for the direct application of lysed blood by pipet. (B) A 5.1 mm double-coated polyester diagnostic tape is stuck on side of 200 &#181;l qPCR tube. The second liner of tape is removed to expose sticky surface for applying capture disk when ready. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54289/54289fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/54289/54289fig3.jpg" /> 
Figure 3: Standard curve of HIV-1 proviral DNA contrived specimens. (A) Amplification plots obtained from 100 &#181;l of 4 replicates HIV-1 negative whole blood spiked with 4,000, 400, 40 and 10 8e5-LAV cells with 500 copies/reaction of the internal control Solid lines = amplification plots; Dotted line = threshold. Y-axis is fluorescence units and X-axis is qPCR cycle number. (B) Standard curves of Cq values calculated from amplification plot versus log copy number of 8e5-LAV cells per 100 &#181;l whole blood. Equation of the line: y = -3.25x + 27.95; R&#178; = 0.996; PCR efficiency = 103.23% calculated via Efficiency = -1+10(-1/slope) <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54289/54289fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/54289/54289fig4.jpg" /> 
Figure 4: Internal control indicates no qPCR inhibition. 500 copies Hydroxypyruvate Reductase Amplification control plasmid added per reaction. Solid lines = amplification plots; Dotted line = threshold. Average Cq of IC = 21.92 &#177; 0.25. Cqs ranged from 21.21 to 22.32. Coefficient of variation (CV %) = 1%; N = 18. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54289/54289fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table fo:keep-together.within-page="1">
	<tbody>
		<tr>
			<td colspan="4">8e5-LAV cells/</td>
		</tr>
		<tr>
			<td>100 &#181;l WB</td>
			<td>Ave. Cq</td>
			<td>SD</td>
			<td>CV (%)</td>
		</tr>
		<tr>
			<td>4,000</td>
			<td>16.27</td>
			<td>0.14</td>
			<td>1</td>
		</tr>
		<tr>
			<td>400</td>
			<td>19.54</td>
			<td>0.15</td>
			<td>1</td>
		</tr>
		<tr>
			<td>40</td>
			<td>22.56</td>
			<td>0.3</td>
			<td>1</td>
		</tr>
		<tr>
			<td>10</td>
			<td>24.83</td>
			<td>0.15</td>
			<td>1</td>
		</tr>
	</tbody>
</table>
Table 1: Average Cq, standard deviation (SD) and coefficient of variation (CV %) of 4,000, 400, 40 and 10 copies of 8e5-LAV standard curve with FINA extraction and HIV-1 specific primers and probes. N = 4. WB = whole blood.

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 ...

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Nanyang Technological University

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JoVE Journal

Biology

18.1K Views.  Northwestern University. 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.

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

Optimization and Comparative Analysis of Plant Organellar DNA Enrichment Methods Suitable for Next-generation Sequencing

Plant organellar genomes contain large, repetitive elements that may undergo pairing or recombination to form complex structures and/or sub-genomic fragments. Organellar genomes also exist in admixtures within a given cell or tissue type (heteroplasmy), and an abundance of subtypes may change throughout development or when under stress (sub-stoichiometric shifting). Next-generation sequencing (NGS) technologies are required to obtain deeper understanding of organellar genome structure and function. Traditional sequencing studies use several methods to obtain organellar DNA: (1) If a large amount of starting tissue is used, it is homogenized and subjected to differential centrifugation and/or gradient purification. (2) If a smaller amount of tissue is used (i.e., if seeds, material, or space is limited), the same process is performed as in (1), followed by whole-genome amplification to obtain sufficient DNA. (3) Bioinformatics analysis can be used to sequence the total genomic DNA and to parse out organellar reads. All these methods have inherent challenges and tradeoffs. In (1), it may be difficult to obtain such a large amount of starting tissue; in (2), whole-genome amplification could introduce a sequencing bias; and in (3), homology between nuclear and organellar genomes could interfere with assembly and analysis. In plants with large nuclear genomes, it is advantageous to enrich for organellar DNA to reduce sequencing costs and sequence complexity for bioinformatics analyses. Here, we compare a traditional differential centrifugation method with a fourth method, an adapted CpG-methyl pulldown approach, to separate the total genomic DNA into nuclear and organellar fractions. Both methods yield sufficient DNA for NGS, DNA that is highly enriched for organellar sequences, albeit at different ratios in mitochondria and chloroplasts. We present the optimization of these methods for wheat leaf tissue and discuss major advantages and disadvantages of each approach in the context of sample input, protocol ease, and downstream application.

The comparison and optimization of two plant organellar DNA enrichment methods are presented: traditional differential centrifugation and fractionation of the total gDNA based on methylation status. We assess the resulting DNA quantity and quality, demonstrate performance in short-read next-generation sequencing, and discuss the potential for use in long-read single-molecule sequencing.

Plant organellar genomes contain large, repetitive elements that may undergo pairing or recombination to form complex structures and/or sub-genomic ...

Genetics

Rapid Fractionation and Isolation of Whole Blood Components in Samples Obtained from a Community-based Setting

Collection and processing of whole blood samples in a non-clinical setting offers a unique opportunity to evaluate community-dwelling individuals both with and without preexisting conditions. Rapid processing of these samples is essential to avoid degradation of key cellular components. Included here are methods for simultaneous peripheral blood mononuclear cell (PBMC), DNA, RNA and serum isolation from a single blood draw performed in the homes of consenting participants across a metropolitan area, with processing initiated within 2 hr of collection. We have used these techniques to process over 1,600 blood specimens yielding consistent, high quality material, which has subsequently been used in successful DNA methylation, genotyping, gene expression and flow cytometry analyses. Some of the methods employed are standard; however, when combined in the described manner, they enable efficient processing of samples from participants of population- and/or community-based studies who would not normally be evaluated in a clinical setting. Therefore, this protocol has the potential to obtain samples (and subsequently data) that are more representative of the general population.

We outline a methodology for the processing of whole blood to obtain a variety of components for further analysis. We have optimized a streamlined protocol that enables rapid, high-throughput simultaneous processing of whole blood samples in a non-clinical setting.

Collection and processing of whole blood samples in a non-clinical setting offers a unique opportunity to evaluate community-dwelling individuals both ...

Medicine

Rapid, Safe, and Simple Manual Bedside Nucleic Acid Extraction for the Detection of Virus in Whole Blood Samples

The rapid diagnosis of an infection is essential for the outbreak management, risk containment, and patient care. We have previously shown a method for the rapid bedside inactivation of the Ebola virus during blood sampling for safe nucleic acid (NA) tests by adding a commercial lysis/binding buffer directly into the vacuum blood collection tubes. Using this bedside inactivation approach, we have developed a safe, rapid, and simplified bedside NA extraction method for the subsequent detection of a virus in lysis/binding buffer-inactivated whole blood. The NA extraction is directly performed in the blood collection tubes and requires no equipment or electricity. 
After the blood is collected into the lysis/binding buffer, the contents are mixed by flipping the tube by hand, and the mixture is incubated for 20 min at room temperature. Magnetic glass particles (MGPs) are added to the tube, and the contents are mixed by flipping the collection tube by hand. The MGPs are then collected on the side of the blood collection tube using a magnetic holder or a magnet and a rubber band. The MGPs are washed three times, and after the addition of elution buffer directly into the collection tube, the NAs are ready for NA tests, such as qPCR or isothermal loop amplification (LAMP), without the removal of the MGPs from the reaction. The NA extraction method is not dependent on any laboratory facilities and can easily be used anywhere (e.g., in field hospitals and hospital isolation wards). When this NA extraction method is combined with LAMP and a portable instrument, a diagnosis can be obtained within 40 min of the blood collection.

Here, we present a protocol for the rapid virus nucleic acid extraction from the virus-inactivated whole blood. The extraction is performed directly in the blood collection tubes and requires no equipment or electricity. The method is not dependent on laboratory facilities and can be used anywhere (e.g., in field hospitals).

The rapid diagnosis of an infection is essential for the outbreak management, risk containment, and patient care. We have previously shown a method for ...

Immunology and Infection

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 ...

Nuclei Isolation from Whole Tissue using a Detergent and Enzyme-Free Method

High-throughput transcriptome and epigenome profiling requires preparation of a single cell or single nuclei suspension. Preparation of the suspension with&#160;intact cell or nuclei involves dissociation and permeabilization, steps that can introduce unwanted noise and undesirable damage. Particularly, certain cell-types such as neurons are challenging to dissociate into individual cells. Additionally, permeabilization of the cellular membrane to release nuclei requires optimization by trial-and-error, which can be time consuming, labor intensive and financially nonviable. To enhance the robustness and reproducibility of sample preparation for high-throughput sequencing, we describe a rapid enzyme and detergent-free column-based nuclei isolation method. The protocol enables efficient isolation of nuclei from the entire zebrafish brain within 20 minutes. The isolated nuclei display intact nuclear morphology and low propensity to aggregate. Further, flow cytometry allows nuclei enrichment and clearance of cellular debris for downstream application. The protocol, which should work on soft tissues and cultured cells, provides a simple and accessible method for sample preparation that can be utilized for high-throughput profiling, simplifying the steps required for successful single-nuclei RNA-seq and ATAC-seq experiments.

Single nuclei isolation relies on dissociation and detergent-based permeabilization of the cell membrane, steps that need optimization and are prone to introducing technical artifacts. We demonstrate a detergent and enzyme-free protocol for rapid isolation of intact nuclei directly from whole tissue, yielding nuclei suitable for single-nucleus RNA-seq (snRNA-Seq) or ATAC-seq.

High-throughput transcriptome and epigenome profiling requires preparation of a single cell or single nuclei suspension. Preparation of the suspension ...

Isolation and Profiling of Single Nuclei from Frozen Mammalian Tissues

A Simple, Quick, and Partially Automated Protocol for the Isolation of Single Nuclei from Frozen Mammalian Tissues for Single Nucleus Sequencing

Single-cell and single-nucleus RNA sequencing have become common laboratory applications due to the wealth of transcriptomic information that they provide. Single nucleus RNA sequencing, particularly, is useful for investigating gene expression in difficult-to-dissociate tissues. Furthermore, this approach is also compatible with frozen (archival) material. Here, we describe a protocol to isolate high-quality single nuclei from frozen mammalian tissues for downstream single nucleus RNA sequencing in a partially-automated manner using commercially available instruments and reagents. Specifically, a robotic dissociator is used to automate and standardize tissue homogenization, followed by an optimized chemical gradient to filter the nuclei. Lastly, we accurately and automatically count the nuclei using an automated fluorescent cell counter. The performance of this protocol is demonstrated on mouse brain, rat kidney, and cynomolgus liver and spleen tissue. This protocol is straightforward, rapid, and readily adaptable to various mammalian tissues without requiring extensive optimization and provides good quality nuclei for downstream single nuclei RNA sequencing.

Author Spotlight: Enhancing Drug Discovery - Development of Automated, Standardized Protocols for Nuclei Extraction from Frozen Tissues 

The study describes a simple, quick, and partially automated protocol to isolate high-quality nuclei from frozen mammalian tissues for downstream single nuclei RNA sequencing.

Single-cell and single-nucleus RNA sequencing have become common laboratory applications due to the wealth of transcriptomic information that they ...

Efficient Technique for Assessing Environmental AMR by Enriching Low-MW DNA

Enhanced Extraction of Low-Molecular Weight DNA from Wastewater for Comprehensive Assessment of Antimicrobial Resistance

Environmental surveillance is recognized as an important tool for assessing public health in the post-pandemic era. Water, in particular wastewater, has emerged as the source of choice to sample pathogen burdens in the environment. Wastewater from open drains and community water treatment plants is a reservoir of both pathogens and antimicrobial resistance (AMR) genes, and frequently comes in contact with humans. While there are many methods of tracking AMR from water,&#160;isolating good-quality DNA at high yields from heterogeneous samples remains a challenge. To compensate, sample volumes often need to be high, creating practical constraints. Additionally, environmental DNA is frequently fragmented, and the sources of AMR (plasmids, phages, linear DNA) consist of low-molecular-weight DNA. Yet, few extraction processes have focused on methods for high-yield extraction of linear and low-molecular-weight DNA. Here, a simple method for high-yield linear DNA extraction from small volumes of wastewater using the precipitation properties of polyethylene glycol (PEG) is reported. This study makes a case for increasing overall DNA yields from water samples collected for metagenomic analyses by enriching the proportion of linear DNA. In addition, enhancing low-molecular-weight DNA overcomes the current problem of under-sampling environmental AMR due to a focus on high-molecular-weight and intracellular DNA. This method is expected to be particularly useful when extracellular DNA exists but at low concentrations, such as with effluents from treatment plants. It should also enhance the environmental sampling of AMR gene fragments that spread through horizontal gene transfer.

Author Spotlight: Understanding and Detecting Environmental Antimicrobial Resistance by Combining Culture-Based Techniques and Genomics

Here, we present a simple technique to assess environmental antimicrobial resistance (AMR) by enhancing the proportion of low-molecular-weight extracellular DNA. Prior treatment with 20%-30% PEG and 1.2 M NaCl allows detection of both genomic and horizontally transferred AMR genes. The protocol lends itself to a kit-free process with additional optimization.

Environmental surveillance is recognized as an important tool for assessing public health in the post-pandemic era. Water, in particular wastewater, has ...

DNA Isolation and Restriction Enzyme Analysis

The revelation of DNA as the hereditary molecule in all organisms has led to enormous scientific and medical breakthroughs and significantly enhanced our understanding of ourselves and other organisms. DNA isolation and profiling have been the fundamental first steps for many of the advancements in the past century; from identification of gene function, to revolutions of agriculture and forensics.

<h4>Isolating DNA from Cells</h4>

DNA isolation is a fairly simple procedure that requires few but essential steps: cell harvesting, lysis, protein degradation, and finally DNA precipitation. Generally, DNA can be harvested from small amounts of tissue, such as a single leaf of a plant, hair follicles of animals, or epithelial cells that can be easily sloughed off within the mouth with a simple saline solution. Since DNA is compartmentalized inside the nucleus or mitochondria within eukaryotic cells, scientists first break apart the cell and nuclear membrane to expose the DNA, which is done with the lysis step. However, once DNA is exposed, it can be degraded by heavy metal ions and extremes of pH. Therefore, a detergent-containing buffer solution is used to not only dissolve the cell membrane, but also to protect DNA from degrading elements in the solution. Moreover, DNA is an unusually long molecule that is condensed inside the nucleus by packing tightly around special proteins called histones. Therefore, an enzyme called Proteinase K, which degrades peptide bonds of proteins, is added to separate the DNA from histones and other proteins. Next, scientists separate DNA from the rest of the cellular extract by using its chemical properties. Due to the negatively charged structure of nucleic acids, Na+ ions in a sodium chloride solution bind to and integrate into entangled DNA strands. Once the DNA is clumped, it can be observed by the naked eye. DNA is insoluble in alcohol and readily precipitates at low temperatures, thus scientists add cold alcohol to precipitate DNA. Finally, the precipitated DNA can be dissolved and stored in deionized water or a mild buffer, free of any enzymes that can degrade nucleic acids, until it is used.

<h4>Quantifying DNA</h4>

Reactions that use DNA often require high purity and precise amounts of DNA. Therefore, following DNA isolation, scientists use a spectrophotometer to quantify the amount of DNA in their sample, as well as the purity of the DNA in the sample. A spectrophotometer measures the intensity of a light beam passed through a sample as a function of its wavelength. Nucleic acids absorb light at 260 nm, whereas proteins absorb light at 280 nm. By measuring the intensity of a light beam at 260 nm, scientists can determine the DNA concentration. Furthermore, by assessing the light intensity at both 260 nm and 280 nm, the purity of the DNA content can be determined. In a sample of pure DNA, the 260/280 nm ratio should approach the value of 2.

<h4>Restriction Enzymes</h4>

For a number of analyses, such as cloning, sequencing, and mapping DNA fragments and genomes, the isolated DNA needs to be cut at specific sequences using restriction enzymes. These enzymes are produced by bacteria as mechanisms of innate immunity against intruding viral DNA and function as molecular scissors. Hence, after restriction enzymes identify a viral DNA, they start to break it apart by cutting it into segments at certain nucleotide sequences they recognize. These nucleotide sequences are often six to twelve nucleotides long and are generally palindromic, which means that they have the same nucleotide sequence in the 3&#8217;-5&#8217; and 5&#8217;-3&#8217; directions. For instance, the restriction enzyme EcoRI cuts DNA at the sequence 3&#8217;...GAATTC&#8230;-5&#8217;, which reads the same in the 5&#8217;-3&#8217; direction. Like EcoRI, there are hundreds of enzymes that correspond to many distinct palindromic sequences. Once DNA is cut by these enzymes, scientists can load the DNA onto a gel in an electrophoresis chamber and pass electrical current through the gel. DNA carries negative charges, which causes DNA fragments to migrate towards the anode. However, the pores of the gel slow down larger DNA fragments relative to smaller DNA sequences, thus the pieces separate according to their size. After the completion of the electrophoresis, DNA fragments can be visualized with the aid of dyes that specifically bind to DNA molecules.

Cutting DNA with restriction enzymes and separating DNA fragments with electrophoresis allows researchers to identify the sizes of unknown fragments relative to a standard ladder. These fragments of interest can then be further isolated and ligated into a plasmid vector for genetic cloning. Genetic engineering and laboratory analyses have been improved immensely with these methods. With DNA profiling, scientists are able to sequence genomes to further understand the workings of organisms and are also able to identify and characterize certain genes1.

DNA isolation and profiling are also practical for events directly impacting everyday life. For example, forensics scientists routinely analyze DNA samples to provide evidence for criminal and civil cases2. Similarly, healthcare workers extract and analyze DNA to check for paternity and genetic diseases. Recently, DNA ancestry analysis kits became popular by allowing users determine their genetic lineage, find relatives, and even discover their genetic disposition to various health conditions3. Finally, with advancements in biotechnology and genetic engineering, personalized medicine is gaining traction among clinical researchers to develop treatments by using an individual&#8217;s genetic data, paving the way to effectively treat patients without detrimental side effects4.

<h4>References</h4>

<ol class="references">
	<li>Chambers, GK, et al. DNA fingerprinting in zoology: past, present, future. Investig Genet. 2014, Vol. 5, 3 doi: 10.1186/2041-2223-5-3.</li>
	<li>Roewer, L. DNA fingerprinting in forensics: past, present, future. Investig Genet. . 2013, Vol. 4, 22 doi: 10.1186/2041-2223-4-22.</li>
	<li>Kirkpatrick, BE and Rashkin, MD. Ancestry Testing and the Practice of Genetic Counseling. J Gen Counseling. 2016, Vol. 26, 1 (6-20).</li>
	<li>Hamburg, MA and Collins, FS. The Path to Personalized Medicine. N Engl J Med . 2010; , Vol. 363, 301-304.</li>
</ol>

The revelation of DNA as the hereditary molecule in all organisms has led to enormous scientific and medical breakthroughs and significantly enhanced our ...

Education

JoVE Lab Manual

Isolating Nucleic Acids from Yeast

One of the many advantages to using yeast as a model system is that large quantities of biomacromolecules, including nucleic acids (DNA and RNA), can be purified from the cultured cells.
This video will address the steps required to carry out nucleic acid extraction. We will begin by briefly outlining the growth and harvest, and lysis of yeast cells, which are the initial steps common to the isolation of all biomacromolecules. Next, we will discuss two unique purification methods for the separation of nucleic acids: column binding and phase separation. Additionally, we will demonstrate several ways in which these methods are applied in the laboratory, including the preparation of nucleic acids for molecular biology techniques such as PCR and southern blotting, quantification of gene expression in response to environmental stimuli, and purification of large amounts of recombinant proteins.

Isolation and Purification of Nucleic Acids from Yeast

One of the many advantages to using yeast as a model system is that large quantities of biomacromolecules, including nucleic acids (DNA and RNA), can be ...

JoVE Science Education

Basic Biology

Biology I: yeast, Drosophila and C. elegans

A Technique for Magnetic Glass Particle-Mediated Nucleic Acid Extraction from Blood

Source: Rosenstierne, M. W., et al. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/58001">Rapid, Safe, and Simple Manual Bedside Nucleic Acid Extraction for the Detection of Virus in Whole Blood Samples.</a> J. Vis. Exp. (2018).
This video demonstrates a technique for nucleic acid extraction from whole human blood using magnetic glass particles (MGPs) &#8212; small magnetic beads coated with silica. Upon lysing the blood cells using a lysis buffer, the released nucleic acids bind to the surface of silica-coated MGPs, extracting the molecules from the sample.

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines
1. Blood Collection from Patients
...

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

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