This research was supported by the Office of Science (BER), U.S. Department of Energy, Cooperative Agreement no. De-FC02-02ER63453, the National Science Foundation&#x2019;s Microbial Genome Sequencing Program (award numbers 0626826 and 0731916 ). We thank John Glass for his technical expertise and advice and K. Eric Wommack for his assistance with environmental sample collection.

1. Preparation of Solutions
Before performing hydroxyapatite chromatography, phosphate buffers must be prepared and the hydroxyapatite must be properly hydrated. 
<ol>
<li>1M Phosphate Solution, pH 6.8: In a 1L flask dissolve 119.98g of sodium phosphate monobasic in 1L of sterile, DEPC-treated H2O. Prepare a 1M sodium phosphate dibasic solution in a 1L flask by dissolving 141.96g of sodium phosphate dibasic in 1L of sterile, DEPC-treated H2O. Combine the mono- and di- solutions at a ratio of 1:1. Place flask on a stir plate and mix using a magnetic stir bar. Adjust pH to 6.8 by adding sodium hydroxide (to increase pH) or phosphoric acid (to decrease pH).</li>
<li> 0.12M Phosphate Buffer: In a 500ml flask, combine 30ml of 1M Phosphate Solution, pH 6.8, 2.5ml of 10% sodium dodecyl sulphate (SDS), and 5ml of 0.5M EDTA. Add sterile, DEPC-treated H2O to a volume of 225ml. Adjust pH to 6.8. Add sterile, DEPC-treated H2O to 250ml. Autoclave.</li> 
<li>0.18M Phosphate Buffer: In a 500ml flask, combine 45ml of 1M Phosphate Solution, pH 6.8, 2.5ml of 10% sodium dodecyl sulphate (SDS), and 5ml of 0.5M EDTA. Add sterile, DEPC-treated H2O to a volume of 225ml. Adjust pH to 6.8. Add sterile, DEPC-treated H2O to 250ml. Autoclave.</li> 
<li> 0.20M Phosphate Buffer: In a 500ml flask, combine 50ml of 1M Phosphate Solution, pH 6.8, 2.5ml of 10% sodium dodecyl sulphate (SDS), and 5ml of 0.5M EDTA. Add sterile, DEPC-treated H2O to a volume of 225ml. Adjust pH to 6.8. Add sterile, DEPC-treated H2O to 250ml. Autoclave.</li> 
<li> 0.40 M Phosphate Buffer: in a 500ml flask, combine 100ml of 1M Phosphate Solution, pH 6.8, 2.5ml of 10% sodium dodecyl sulphate (SDS), and 5ml of 0.5M EDTA. Add sterile, DEPC-treated H2O to a volume of 225ml. Adjust pH to 6.8. Add sterile, DEPC-treated H2O to 250ml. Autoclave.</li> 
<li>&#x201C;1.00M Phosphate Buffer&#x201D; (actual concentration of phosphate in this solution is .91M): In a 500ml flask, combine 30ml of 1M Phosphate Solution, pH6.8, 2.5ml of 10% sodium dodecyl sulphate (SDS), and 5ml of 0.5M EDTA. Add sterile, DEPC-treated H2O to a volume of 225ml. Adjust pH to 6.8. Add sterile, DEPC-treated H2O to 250ml. Autoclave.</li> 
<li>Hydroxyapatite hydration: Weight out 1g of hydroxyapatite and add to a 50ml tube. Add 6ml of 0.12M phosphate buffer to the hydroxyapatite and invert to mix. Prior to use bring temperature to 60&deg;C and mix thoroughly. Store for extended periods of time at 4&deg;C.</li>
<li>Prior to use, equilibrate all phosphate buffers and hydrated hydroxyapatite at 60&deg;C.</li> 
</ol>
2. Preparation of Econo-Column
<ol>
<li>Close stopcock. Rinse column several times with deionized H2O by repeatedly inverting the column and decanting.</li>
<li> Remove the stopcock from the Econo-column and autoclave the column to sterilize.</li>
<li> Replace and close stopcock. Add 1ml of Sigmacote to the sterile Econo-column and coat all glass surfaces. Open the stopcock and decant.</li>
<li> Attach column to a circulating water bath and set temperature to 60&deg;C. To einsure the least amount of heat loss across the column wrapping the column in an insulating agent such as aluminum foil or foam pipe insulation is recommended.</li> 
<li> Rinse the Econo-column twice with sterile, RNase-free H2O then twice with sterile, RNase-free 0.12M Phosphate Buffer.</li>
</ol>
3. Hydroxypaptite Chromotography
<ol>
<li> Making sure the stopcock is closed, slowly add 2ml of pre-prepared, resuspended hydroxyapatite to the bottom of the Econo-column using a sterile 2ml serological pipette.</li>
<li> Allow the hydroxyapatite to settle under the force of gravity for 30 minutes.</li>
<li> Drain buffer from column and close stopcock. Combine nucleic acids with 0.12M phosphate buffer in a final volume of 500 &mu;l and incubate at 60&deg;C for 10 minutes in a heat block or water bath.</li> 
<li> Quickly apply nucleic acids prepared in 0.12M phosphate buffer to the hydroxyapatite using a 2ml serological pipette making sure not to disturb the column.</li>
<li> Allow the nucleic acids to bind to the hydroxyapatite for 30 minutes.</li>
<li> Place a 15ml tube under the column, open the stopcock and collect the initial sample containing ssDNA. Close stopcock.</li>
<li> Add 6ml of 0.12M phosphate buffer to the hydroxyapatite making sure not to disturb the column, open the stopcock, and collect single-stranded DNA in the 15ml tube from the previous step. Close stopcock.</li>
<li> Add 1 volume of a phenol:chloroform:isoamyl alcohol (25:25:1 v/v/v) solution to the tube, mix vigorously and centrifuge at 3500 x g for 15 minutes. Transfer supernatant containing ssDNA to a new 15ml tube.</li>
<li> Repeat 3.76 and 3.87 using 6ml of 0.20M phosphate buffer to elute and purify RNA from the column making sure to use a new 15ml tube for collection.</li>
<li>Repeat 3.76 and 3.87 using 6ml of 0.40M phosphate buffer to elute and purify dsDNA from the column making sure to use a new 15ml tube for collection. </li> 
<li> Repeat 3.76 and 3.87 using 6ml of 1.00M phosphate buffer to strip the column of any residual dsDNA making sure to use a new 15ml tube for collection.</li>
</ol>
4. Desalting Nucleic Acid Samples
<ol>
<li>Transfer 4-5ml of sample to an Amicon Ultra-4 centrifugal filter device outfitted with a 30,000 molecular weight cut off Ultracel membrane.</li>
<li>Concentrate sample to &lt; 500&mu;l by spinning at 6,000 x g, 30&deg;C, 5 minutes (or until concentrated volume is achieved) in a fixed angle rotor. Discard flow through.</li>
<li> Add the remainder of the sample(s) to the Amicon Ultra-4 centrifugal filter device and bring the volume up to 4-5ml with Rnase free 1X TE Buffer.</li> 
<li> Concentrate sample to &lt; 500&mu;l by spinning at 6,000 x g, 30&deg;C, 5 minutes (or until concentrated volume is achieved) in a fixed angle rotor. Discard flow through.</li>
<li>Add 4-5ml of Rnase free 1X TE Buffer to the Amicon Ultra-4 centrifugal filter device. Concentrate sample to &lt; 500&mu;l by centrifuging at 6,000 x g, 30&deg;C, 5 minutes (or until concentrated volume is achieved) in a fixed angle rotor. Discard flow through.</li>
<li> Repeat step 4.5 an additional 5 times to completely desalt nucleic acid sample(s).</li>
<li>Transfer remaining sample(s) to a sterile 1.7ml eppendorf tube. Add 1/10th volume 3M sodium acetate, pH 7.0, two volumes of 100% ethanol, and 1&mu;l of Glycoblue. Mix well by vortexing.</li>
<li>Centrifuge at 28,000 x g, 4&deg;C, 60 minutes. Decant making sure not to disturb the pellet. Add 300&mu;l of 70% ethanol. Spin at 28,000 x g, room temperature for 10 minutes. Decant, dry and resuspend each fraction in an appropriate volume of Rnase-free TE buffer.</li>
</ol>
5. Representative Results:
Figure 1 summarizes the methods presented for using hydroxyapatite chromatography to fractionate ssDNA, dsDNA and RNA nucleic acids from a mixed viral assemblage. This method exploits the charge interaction between the negatively charged phosphate backbone of the nucleic acids and the positively charged Ca2+ ions present in the hydroxyapatite and allows for the efficient fractionation of nucleic acid subtypes (ssDNA, dsDNA and RNA) with increasing phosphate buffer concentrations 12. 
The hydroxyapatite fractionation of an in vitro &#x201C;community&#x201D; of known ssDNA (M13mp18), dsDNA (lambda) and RNA (MS2 and phi6) viral genomes is illustrated in Figure 2. The nucleic acids were combined in equal concentrations, applied to the hydroxyapatite column and were eluted using an increasing concentration of phosphate buffer. Each nucleic acid subtype elutes independent of the others with less than 9% carry-over from one fraction to the next.
The application of this technique to nucleic acids isolated from a viral community collected from the Chesapeake Bay is shown in Figure 3. The total nucleic acid yield isolated from purified viruses was applied to the hydroxyapatite column. Genomic material consisting of ssDNA, RNA and dsDNA was independently eluted with 0.12M, 0.20M and 0.40M/1.00M concentrations of phosphate buffer respectively. Double-stranded DNA is eluted at phosphate concentrations of 0.40M and 1.00M and in this case, dominates this viral community compared to ssDNA and RNA genotypes. This observation is consistent with the expected viral community composition of marine and estuarine environments where the majority of recoverable nucleic acids are dsDNA 13.
<img src="/files/ftp_upload/3146/3146fig1.jpg" alt="Figure 1"/> Figure 1. Flow diagram of the hydroxyapatite chromatography method for the fractionation of nucleic acids. A mixture of ssDNA, dsDNA and RNA prepared in 0.12M phosphate buffer is heated to 60&deg;C and applied to a hydroxyapatite column maintained at a constant 60&deg;C using a circulating water bath. Nucleic acids (ssDNA, RNA and dsDNA) are eluted from the hydroxyapatite with increasing concentrations of a phosphate-containing buffer. This figure has been reproduced with permission from American Society of Microbiology and was originally featured in Andrews-Pfannkoch et al. 201012.
<img src="/files/ftp_upload/3146/3146fig2.jpg" alt="Figure 2"/> Figure 2. Separation of known viral nucleic acids using hydroxyapatite chromatography. Equal concentrations of M13mp18 ssDNA, MS2 ssRNA, phi6 dsRNA and lambda dsDNA (lanes 2-5, respectively) were combined (lane 6) and applied to a hydroxyapatite column. Single-stranded DNA (M13mp18), RNA (MS2/phi6) and dsDNA (lambda) were independently eluted from the hydroxyapatite column using 0.12M (lane 8), 0.18M (lane 9) and 0.40M/1.00M (lane 10). A 1kb ladder (lanes 1, 7) was used to confirm genome sizes. This figure has been reproduced with permission from American Society of Microbiology and was originally featured in Andrews-Pfannkoch et al. 201012.
<img src="/files/ftp_upload/3146/3146fig3.jpg" alt="Figure 3"/> Figure 3. Separation of viral nucleic acids isolated from a community within the Chesapeake Bay. Nucleic acids were isolated and applied to a hydroxyapatite column. ssDNA(lane 0.12M), RNA (lane 0.20M) and dsDNA(lanes 0.40M/1.0M) were independently eluted using phosphate buffer concentrations of 0.12M, 0.20M and 0.40M/1.00M, respectively. Hi Mass DNA Ladder (lane L1) and 1kb Ladder (lane L2) were used to visually confirm approximate molecular weight and mass of nucleic acids. This figure has been reproduced with permission from American Society of Microbiology and was originally featured in Andrews-Pfannkoch et al. 201012.

<ol>
	<li>Whitman, W. B., Coleman, D. C., Wiebe, W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prokaryotes:+The+Unseen+Majority.">Prokaryotes: The Unseen Majority.</a> Proceedings of the National Academy of Sciences of the United States of America. 95, 6578-65 (1998).</li><li>Hendrix, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacteriophages:+evolution+of+the+majority.">Bacteriophages: evolution of the majority.</a> Theor Popul Biol. 61, 471-471 (2002).</li><li>Weinbauer, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacteriophages:+Evolution+of+the+Majority.">Bacteriophages: Evolution of the Majority.</a> FEMS Microbiol Rev. 28, 127-127 (2004).</li><li>Dinsdale, E. A., Edwards, R. A., Hall, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+Metabolic+Profiling+of+Nine+Biomes.">Functional Metabolic Profiling of Nine Biomes.</a> Nature. 455, 830-830 (2008).</li><li>McDaniel, L., Breitbart, M., Mobberley, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metagenomic+Analysis+of+Lysogeny+in+Tampa+Bay:+Implications+for+Prophage+Gene+Expression.">Metagenomic Analysis of Lysogeny in Tampa Bay: Implications for Prophage Gene Expression.</a> PLoS ONE. 3, e3263-e3263 (2008).</li><li>Williamson, S. J., Rusch, D. B., Yooseph, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Sorcerer+II+Global+Ocean+Sampling+Expedition:+Metagenomic+Characterization+of+Viruses+within+Aquatic+Microbial+Samples.">The Sorcerer II Global Ocean Sampling Expedition: Metagenomic Characterization of Viruses within Aquatic Microbial Samples.</a> PLoS ONE. 3, e1456-e1456 (2008).</li><li>Lang, A. S., Rise, M. L., Culley, A. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA+Viruses+in+the+Sea.">RNA Viruses in the Sea.</a> FEMS Microbiol Rev. 33, 295-295 (2009).</li><li>Ng, T. F., Manire, C., Borrowman, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Discovery+of+a+Novel+Single-Stranded+DNA+Virus+from+a+Sea+Turtle+Fibropapilloma+by+using+Viral+Metagenomics.">Discovery of a Novel Single-Stranded DNA Virus from a Sea Turtle Fibropapilloma by using Viral Metagenomics.</a> J. Virol. 83, 2500-2500 (2009).</li><li>Rosario, K., Duffy, S., Breitbart, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diverse+Circovirus-like+Genome+Architectures+Revealed+by+Environmental+Metagenomics.">Diverse Circovirus-like Genome Architectures Revealed by Environmental Metagenomics.</a> J Gen Virol. 90, 2418-2418 (2009).</li><li>Culley, A. I., Lang, A. S., Suttle, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metagenomic+Analysis+of+Coastal+RNA+Virus+Communities.">Metagenomic Analysis of Coastal RNA Virus Communities.</a> Science. 312, 1795-1795 (2006).</li><li>Bernardi, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromotography+of+Nucleic+Acids+on+Hydroxyapatite.">Chromotography of Nucleic Acids on Hydroxyapatite.</a> Nature. 209, 779-779 (1965).</li><li>Andrews-Pfannkoch, C., Fadrosh, D. W., Thorpe, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydroxyapatite-Mediated+Separation+of+Double-Stranded+DNA,+Single-Stranded+DNA+and+RNA+Genomes+from+Natural+Viral+Assemblages.">Hydroxyapatite-Mediated Separation of Double-Stranded DNA, Single-Stranded DNA and RNA Genomes from Natural Viral Assemblages.</a> Applied and environmental microbiology. 76, 5039-5039 (2010).</li><li>Wommack, K. E., Colwell, R. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Virioplankton:+Viruses+in+Aquatic+Ecosystems.">Virioplankton: Viruses in Aquatic Ecosystems.</a> Microbiol Mol Biol Rev. 64, 69-69 (2000).</li></ol>

No conflicts of interest declared.

The hydroxyapatite chromatography methodology presented here is a highly efficient and robust tool for the fractionation of nucleic acids from mixed viral assemblages, when the goal is to study the total nucleic acid composition of the community. Generally, ssDNA, RNA and dsDNA will elute from the column pho phosphate buffer concentrations greater than approximately&tilde; 0and 0.40M respectively. However, each preparation of hydroxyapatite can have a slightly different composition and elution profile so it ...

<table border="1">
	<tbody>
		<tr>
			<th>Material Name</th>
			<th>Company</th>
			<th>Catalogue Number</th>
			<th>Catalogue Number</th>
		</tr>
		<tr>
			<td>Econo-Column</td>
			<td>Bio-Rad</td>
			<td>737-0717</td>
			<td>0.7cm ID package/2</td>
		</tr>
		<tr>
			<td>Hydroxyapatite</td>
			<td>Bio-Rad</td>
			<td>130-0520</td>
			<td>DNA Grade Bio-Gel HTP Gel, 100g/td>
		</tr>
		<tr>
			<td>Sodium Phosphate, Monobasic</td>
			<td>VWR</td>
			<td>VW1497-01</td>
			<td>Monohydrate, Crystal 500g</td>
		</tr>
		<tr>
			<td>Sodium Phosphate, Dibasic</td>
			<td>VWR</td>
			<td>VW1496-01</td>
			<td>Anhydrous, Powder 500g</td>
		</tr>
		<tr>
			<td>DEPC-treated Water</td>
			<td>Invitrogen</td>
			<td>AM9922</td>
			<td>1L</td>
		</tr>
		<tr>
			<td>10% SDS Solution</td>
			<td>Invitrogen</td>
			<td>24730-020</td>
			<td>UltraPure, 1L</td>
		</tr>
		<tr>
			<td>0.5M EDTA</td>
			<td>Invitrogen</td>
			<td>AM9262</td>
			<td>pH 8.0, 1L</td>
		</tr>
		<tr>
			<td>Sigmacote</td>
			<td>Sigma-Aldrich</td>
			<td>SL2-25ML</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>2ml serological pippette</td>
			<td>VWR</td>
			<td>89130-884</td>
			<td>Polystyrene, Sterile</td>
		</tr>
		<tr>
			<td>BD Falcon Centrifuge Tubes</td>
			<td>VWR</td>
			<td>21008-936</td>
			<td>15ml, Sterile</td>
		</tr>
		<tr>
			<td>Phenol:Chloroform:Isoamyl Alcohol (25:24:1 v/v/v)</td>
			<td>Invitrogen</td>
			<td>15593-031</td>
			<td>UltraPure, 100ml</td>
		</tr>
		<tr>
			<td>Amicon Ultra-4 Centrifugal Device</td>
			<td>Millipore</td>
			<td>UFC803024</td>
			<td>Ultracel-30 membrane</td>
		</tr>
		<tr>
			<td>20X TE Buffer, Rnase free</td>
			<td>Invitrogen</td>
			<td>T11493</td>
			<td>100ml</td>
		</tr>
		<tr>
			<td>Glycoblue</td>
			<td>Invitrogen</td>
			<td>AM9516</td>
			<td>15mg/ml</td>
		</tr>
	</tbody>
</table>

separation of single-stranded dna, double-stranded dna and rna from an environmental viral community using hydroxyapatite chromatography

Viruses, particularly bacteriophages (phages), are the most numerous biological entities on Earth1,2. Viruses modulate host cell abundance and diversity, contribute to the cycling of nutrients, alter host cell phenotype, and influence the evolution of both host cell and viral communities through the lateral transfer of genes 3. Numerous studies have highlighted the staggering genetic diversity of viruses and their functional potential in a variety of natural environments. 
Metagenomic techniques have been used to study the taxonomic diversity and functional potential of complex viral assemblages whose members contain single-stranded DNA (ssDNA), double-stranded DNA (dsDNA) and RNA genotypes 4-9. Current library construction protocols used to study environmental DNA-containing or RNA-containing viruses require an initial nuclease treatment in order to remove nontargeted templates 10. However, a comprehensive understanding of the collective gene complement of the virus community and virus diversity requires knowledge of all members regardless of genome composition. Fractionation of purified nucleic acid subtypes provides an effective mechanism by which to study viral assemblages without sacrificing a subset of the community&#x2019;s genetic signature. 
Hydroxyapatite, a crystalline form of calcium phosphate, has been employed in the separation of nucleic acids, as well as proteins and microbes, since the 1960s11. By exploiting the charge interaction between the positively-charged Ca2+ ions of the hydroxyapatite and the negatively charged phosphate backbone of the nucleic acid subtypes, it is possible to preferentially elute each nucleic acid subtype independent of the others. We recently employed this strategy to independently fractionate the genomes of ssDNA, dsDNA and RNA-containing viruses in preparation of DNA sequencing 12. Here, we present a method for the fractionation and recovery of ssDNA, dsDNA and RNA viral nucleic acids from mixed viral assemblages using hydroxyapatite chromotography.

Watch this Scientific Journal Video about Separation of Single-stranded DNA, Double-stranded DNA and RNA from an Environmental Viral Community Using Hydroxyapatite Chromatography at JoVE.com

Separation of Single-stranded DNA, Double-stranded DNA and RNA from an Environmental Viral Community Using Hydroxyapatite Chromatography

We describe an efficient method to separate single-stranded DNA, double-stranded DNA and RNA molecules from environmental viral communities. Nucleic acids are fractionated using hydroxyapatite chromatography with increasing concentrations of phosphate-containing buffers. This method permits the isolation of all viral nucleic acid types from environmental samples.

Viruses, particularly bacteriophages (phages), are the most numerous biological entities on Earth1,2.  Viruses modulate host cell abundance and diversity, ...

separation-single-stranded-dna-double-stranded-dna-rna-from-an

Research

JoVE Journal

Immunology and Infection

31.5K 조회수.  The J. Craig Venter Institute. 우리는 환경 바이러스성 사회에서 단일 좌초 DNA를 두 번 좌초 DNA와 RNA 분자를 분리하는 효율적인 방법을 설명합니다. 핵산은 인산 함유 버퍼의 농도 증가와 히드록 시아파 타이트 크로마 토그래피를 사용하여 fractionated 있습니다. 이 방법은 환경 시료에서 바이러스 핵산 형식의 절연을 허용합니다.

비디오: Separation of Single-stranded DNA, Double-stranded DNA and RNA from an Environmental Viral Community Using Hydroxyapatite Chromatography

Generation of Recombinant Influenza Virus from Plasmid DNA

Efforts by a number of influenza research groups have been pivotal in the development and improvement of influenza A virus reverse genetics. Originally established in 1999 1,2 plasmid-based reverse genetic techniques to generate recombinant viruses have revolutionized the influenza research field because specific questions have been answered by genetically engineered, infectious, recombinant influenza viruses. Such studies include virus replication, function of viral proteins, the contribution of specific mutations in viral proteins in viral replication and/or pathogenesis and, also, viral vectors using recombinant influenza viruses expressing foreign proteins 3.

Rescue of influenza A viruses from plasmid DNA is a basic and essential experimental technique that allows influenza researchers to generate recombinant viruses to study multiple aspects in the biology of influenza virus, and to be used as potential vectors or vaccines.

Efforts by a number of influenza research groups have been pivotal in the development and improvement of influenza A virus reverse genetics. Originally ...

Isolation of Fidelity Variants of RNA Viruses and Characterization of Virus Mutation Frequency

RNA viruses use RNA dependent RNA polymerases to replicate their genomes. The intrinsically high error rate of these enzymes is a large contributor to the generation of extreme population diversity that facilitates virus adaptation and evolution. Increasing evidence shows that the intrinsic error rates, and the resulting mutation frequencies, of RNA viruses can be modulated by subtle amino acid changes to the viral polymerase. Although biochemical assays exist for some viral RNA polymerases that permit quantitative measure of incorporation fidelity, here we describe a simple method of measuring mutation frequencies of RNA viruses that has proven to be as accurate as biochemical approaches in identifying fidelity altering mutations. The approach uses conventional virological and sequencing techniques that can be performed in most biology laboratories. Based on our experience with a number of different viruses, we have identified the key steps that must be optimized to increase the likelihood of isolating fidelity variants and generating data of statistical significance. The isolation and characterization of fidelity altering mutations can provide new insights into polymerase structure and function1-3. Furthermore, these fidelity variants can be useful tools in characterizing mechanisms of virus adaptation and evolution4-7.

The present article describes the steps required to isolate and characterize RNA polymerase fidelity variants of RNA viruses and how to use mutation frequency data to confirm fidelity changes in tissue culture.

RNA viruses use RNA dependent RNA polymerases to replicate their genomes. The intrinsically high error rate of these enzymes is a large contributor to the ...

Development of Cell-type specific anti-HIV gp120 aptamers for siRNA delivery

The global epidemic of infection by HIV has created an urgent need for new classes of antiretroviral agents. The potent ability of small interfering (si)RNAs to inhibit the expression of complementary RNA transcripts is being exploited as a new class of therapeutics for a variety of diseases including HIV. Many previous reports have shown that novel RNAi-based anti-HIV/AIDS therapeutic strategies have considerable promise; however, a key obstacle to the successful therapeutic application and clinical translation of siRNAs is efficient delivery. Particularly, considering the safety and efficacy of RNAi-based therapeutics, it is highly desirable to develop a targeted intracellular siRNA delivery approach to specific cell populations or tissues. The HIV-1 gp120 protein, a glycoprotein envelope on the surface of HIV-1, plays an important role in viral entry into CD4 cells. The interaction of gp120 and CD4 that triggers HIV-1 entry and initiates cell fusion has been validated as a clinically relevant anti-viral strategy for drug discovery. 
Herein, we firstly discuss the selection and identification of 2'-F modified anti-HIV gp120 RNA aptamers. Using a conventional nitrocellulose filter SELEX method, several new aptamers with nanomolar affinity were isolated from a 50 random nt RNA library. In order to successfully obtain bound species with higher affinity, the selection stringency is carefully controlled by adjusting the conditions. The selected aptamers can specifically bind and be rapidly internalized into cells expressing the HIV-1 envelope protein. Additionally, the aptamers alone can neutralize HIV-1 infectivity. Based upon the best aptamer A-1, we also create a novel dual inhibitory function anti-gp120 aptamer-siRNA chimera in which both the aptamer and the siRNA portions have potent anti-HIV activities. Further, we utilize the gp120 aptamer-siRNA chimeras for cell-type specific delivery of the siRNA into HIV-1 infected cells. This dual function chimera shows considerable potential for combining various nucleic acid therapeutic agents (aptamer and siRNA) in suppressing HIV-1 infection, making the aptamer-siRNA chimeras attractive therapeutic candidates for patients failing highly active antiretroviral therapy (HAART).

Several 2&#x2019;-Fluoro RNA aptamers against HIV-1Ba-L gp120 with nanomole affinity are isolated from a RNA library by in vitro SELEX procedure. A new dual inhibitory function anti-gp120 aptamer-siRNA chimera is created and shows considerable promise for systemic anti-HIV therapy.

The global epidemic of infection by HIV has created an urgent need for new classes of antiretroviral agents. The potent ability of small interfering ...

Preparation of Viral DNA from Nucleocapsids

Viruses are obligate cellular parasites, and thus the study of their DNA requires isolating viral material away from host cell contaminants and DNA. Several downstream applications require large quantities of pure viral DNA, which is provided by this protocol. These applications include viral genome sequencing, where the removal of host DNA is crucial to optimize data output for viral sequences, and the production of new viral recombinant strains, where co-transfection of purified plasmid and linear viral DNA facilitates recombination.1,2,3 
This procedure utilizes a combination of extractions and density-based centrifugation to isolate purified linear herpesvirus nucleocapsid DNA from infected cells.4,5 The initial purification steps aim to isolate purified viral capsids, which contain and protect the viral DNA during the extractions and centrifugation steps that remove cellular proteins and DNA. Lysis of nucleocapsids then releases viral DNA, and two final phenol-chloroform steps remove remaining proteins. The final DNA captured from solution is highly concentrated and pure, with an average OD260/280 of 1.90. Depending on the quantity of infected cells used, yields of viral DNA range from 150-800 &mu;g or more. The purity of this DNA makes it stable during long-term storage at 4C. This DNA is thus ideally suited for high-throughput sequencing, high fidelity PCR reactions, and transfections. 
Prior to beginning the protocol, it is important to know the average number of cells per dish (e.g. an average of 8 x 106 PK-15 cells in a confluent 15 cm dish), and the titer of the viral stock to be used (e.g. 1 x 108 plaque-forming units per ml). These are necessary to calculate the appropriate multiplicity of infection (MOI) for the protocol.6 For instance, to infect one 15 cm dish of PK-15 cells with the above viral stock, at an MOI of 5, you would use 400 &mu;l of viral stock and dilute it with 3.6 ml of medium (total inoculation volume of 4 ml for one 15 cm plate).
Multiple viral DNA preparations can be prepared at the same time. The number of simultaneous preparations is limited only by the number of tubes held by the ultracentrifuge rotor (one per virus; see step 3.9 below). Here we describe the procedure as though being done for one virus.

We describe the process of isolating high purity herpesvirus nucleocapsid DNA from infected cells. The final DNA captured from solution is of high concentration and purity, making it ideally suited for high-throughput sequencing, high fidelity PCR reactions, and transfections to produce new viral recombinants.

Viruses are obligate cellular parasites, and thus the study of their DNA requires isolating viral material away from host cell contaminants and DNA. ...

A Simple Chelex Protocol for DNA Extraction from Anopheles spp.

Endemic countries are increasingly adopting molecular tools for efficient typing, identification and surveillance against malaria parasites and vector mosquitoes, as an integral part of their control programs1,2,3,4,5. For sustainable establishment of these accurate approaches in operations research to strengthen malaria control and elimination efforts, simple and affordable methods, with parsimonious reagent and equipment requirements are essential6,7,8. Here we present a simple Chelex-based technique for extracting malaria parasite and vector DNA from field collected mosquito specimens.
We morphologically identified 72 Anopheles gambiae sl. from 156 mosquitoes captured by pyrethrum spray catches in sleeping rooms of households within a 2,000 km2 vicinity of the Malaria Institute at Macha. After dissection to separate the head and thorax from the abdomen for all 72 Anopheles gambiae sl. mosquitoes, the two sections were individually placed in 1.5 ml microcentrifuge tubes and submerged in 20 &mu;l of deionized water. Using a sterile pipette tip, each mosquito section was separately homogenized to a uniform suspension in the deionized water. Of the ensuing homogenate from each mosquito section, 10 &mu;l was retained while the other 10 &mu;l was transferred to a separate autoclaved 1.5 ml tube. The separate aliquots were subjected to DNA extraction by either the simplified Chelex or the standard salting out extraction protocol9,10. The salting out protocol is so-called and widely used because it employs high salt concentrations in lieu of hazardous organic solvents (such as phenol and chloroform) for the protein precipitation step during DNA extraction9.
Extracts were used as templates for PCR amplification using primers targeting arthropod mitochondrial nicotinamide adenine dinucleotide dehydrogenase (NADH) subunit 4 gene (ND4) to check DNA quality11, a PCR for identification of Anopheles gambiae sibling species10 and a nested PCR for typing of Plasmodium falciparum infection12. Comparison using DNA quality (ND4) PCR showed 93% sensitivity and 82% specificity for the Chelex approach relative to the established salting out protocol. Corresponding values of sensitivity and specificity were 100% and 78%, respectively, using sibling species identification PCR and 92% and 80%, respectively for P. falciparum detection PCR. There were no significant differences in proportion of samples giving amplicon signal with the Chelex or the regular salting out protocol across all three PCR applications. The Chelex approach required three simple reagents and 37 min to complete, while the salting out protocol entailed 10 different reagents and 2 hr and 47 min' processing time, including an overnight step. Our results show that the Chelex method is comparable to the existing salting out extraction and can be substituted as a simple and sustainable approach in resource-limited settings where a constant reagent supply chain is often difficult to maintain.

A Simple Chelex Protocol for DNA Extraction from Anopheles spp.

A rapid and affordable way to extract quality malaria parasite and vector DNA from mosquito specimens is described. Capitalizing on chelating properties of Chelex resin, the simple method enables genotyping of malaria parasites in mosquito mid-gut and salivary gland phases, as well as molecular identification of the Anopheles sibling species by PCR.

Endemic countries are increasingly adopting molecular tools for  efficient typing, identification and surveillance against malaria parasites and  vector ...

Establishment of an In vitro System to Study Intracellular Behavior of Candida glabrata in Human THP-1 Macrophages

A cell culture model system, if a close mimic of host environmental conditions, can serve as an inexpensive, reproducible and easily manipulatable alternative to animal model systems for the study of a specific step of microbial pathogen infection. A human monocytic cell line THP-1 which, upon phorbol ester treatment, is differentiated into macrophages, has previously been used to study virulence strategies of many intracellular pathogens including Mycobacterium tuberculosis. Here, we discuss a protocol to enact an in vitro cell culture model system using THP-1 macrophages to delineate the interaction of an opportunistic human yeast pathogen Candida glabrata with host phagocytic cells. This model system is simple, fast, amenable to high-throughput mutant screens, and requires no sophisticated equipment. A typical THP-1 macrophage infection experiment takes approximately 24 hr with an additional 24-48 hr to allow recovered intracellular yeast to grow on rich medium for colony forming unit-based viability analysis. Like other in vitro model systems, a possible limitation of this approach is difficulty in extrapolating the results obtained to a highly complex immune cell circuitry existing in the human host. However, despite this, the current protocol is very useful to elucidate the strategies that a fungal pathogen may employ to evade/counteract antimicrobial response and survive, adapt, and proliferate in the nutrient-poor environment of host immune cells.

Establishment of an In vitro System to Study Intracellular Behavior of Candida glabrata in Human THP-1 Macrophages

The current article outlines a protocol to establish an in vitro cell culture model system to study the interaction of a facultative intracellular human fungal pathogen Candida glabrata with human macrophages which will be a useful tool to advance our knowledge of fungal virulence mechanisms.

A cell culture model system, if a close mimic of host environmental conditions, can serve as an inexpensive, reproducible and easily manipulatable ...

Using Click Chemistry to Measure the Effect of Viral Infection on Host-Cell RNA Synthesis

Many RNA viruses have evolved the ability to inhibit host cell transcription as a means to circumvent cellular defenses. For the study of these viruses, it is therefore important to have a quick and reliable way of measuring transcriptional activity in infected cells. Traditionally, transcription has been measured either by incorporation of radioactive nucleosides such as 3H-uridine followed by detection via autoradiography or scintillation counting, or incorporation of halogenated uridine analogs such as 5-bromouridine (BrU) followed by detection via immunostaining. The use of radioactive isotopes, however, requires specialized equipment and is not feasible in a number of laboratory settings, while the detection of BrU can be cumbersome and may suffer from low sensitivity.
The recently developed click chemistry, which involves a copper-catalyzed triazole formation from an azide and an alkyne, now provides a rapid and highly sensitive alternative to these two methods. Click chemistry is a two step process in which nascent RNA is first labeled by incorporation of the uridine analog 5-ethynyluridine (EU), followed by detection of the label with a fluorescent azide. These azides are available as several different fluorophores, allowing for a wide range of options for visualization.
This protocol describes a method to measure transcriptional suppression in cells infected with the Rift Valley fever virus (RVFV) strain MP-12 using click chemistry. Concurrently, expression of viral proteins in these cells is determined by classical intracellular immunostaining. Steps 1 through 4 detail a method to visualize transcriptional suppression via fluorescence microscopy, while steps 5 through 8 detail a method to quantify transcriptional suppression via flow cytometry. This protocol is easily adaptable for use with other viruses.

This method describes the use of click chemistry to measure changes in host cell transcription after infection with the Rift Valley fever virus (RVFV) strain MP-12. Results can be visualized qualitatively via fluorescence microscopy or obtained quantitatively through flow cytometry. This method is adaptable for use with other viruses.

Many RNA viruses have evolved the ability to inhibit host cell transcription as a means to circumvent cellular defenses. For the study of these viruses, ...

Averaging of Viral Envelope Glycoprotein Spikes from Electron Cryotomography Reconstructions using Jsubtomo

Enveloped viruses utilize membrane glycoproteins on their surface to mediate entry into host cells. Three-dimensional structural analysis of these glycoprotein &#8216;spikes&#8217; is often technically challenging but important for understanding viral pathogenesis and in drug design. Here, a protocol is presented for viral spike structure determination through computational averaging of electron cryo-tomography data. Electron cryo-tomography is a technique in electron microscopy used to derive three-dimensional tomographic volume reconstructions, or tomograms, of pleomorphic biological specimens such as membrane viruses in a near-native, frozen-hydrated state. These tomograms reveal structures of interest in three dimensions, albeit at low resolution. Computational averaging of sub-volumes, or sub-tomograms, is necessary to obtain higher resolution detail of repeating structural motifs, such as viral glycoprotein spikes. A detailed computational approach for aligning and averaging sub-tomograms using the Jsubtomo software package is outlined. This approach enables visualization of the structure of viral glycoprotein spikes to a resolution in the range of 20-40 &#197; and study of the study of higher order spike-to-spike interactions on the virion membrane. Typical results are presented for Bunyamwera virus, an enveloped virus from the family Bunyaviridae. This family is a structurally diverse group of pathogens posing a threat to human and animal health.

An approach is presented for determining structures of viral membrane glycoprotein complexes using a combination of electron cryo-tomography and sub-tomogram averaging with the computational package Jsubtomo.

Enveloped viruses utilize membrane glycoproteins on their surface to mediate entry into host cells. Three-dimensional structural analysis of these ...

Isolation of Infiltrating Leukocytes from Mouse Skin Using Enzymatic Digest and Gradient Separation

Dissociating murine skin into a single cell suspension is essential for downstream cellular analysis such as the characterization of infiltrating immune cells in rodent models of skin inflammation. Here, we describe a protocol for the digestion of mouse skin in a nutrient-rich solution with collagenase D, followed by separation of hematopoietic cells using a discontinuous density gradient. Cells thus obtained can be used for in vitro studies, in vivo transfer, and other downstream cellular and molecular analyses including flow cytometry. This protocol is an effective and economical alternative to expensive mechanical dissociators, specialized separation columns, and harsher trypsin- and dispase-based digestion methods, which may compromise cellular viability or density of surface proteins relevant for phenotypic characterization or cellular function. As shown here in our representative data, this protocol produced highly viable cells, contained specific immune cell subsets, and had no effect on integrity of common surface marker proteins used in flow cytometric analysis.

This protocol describes enzymatic digestion of mouse skin in nutrient-rich medium followed by gradient separation to isolate leukocytes. Cells thus derived can be used for diverse downstream applications. This is an effective, economical, and improved alternative to tissue dissociation machines and harsher trypsin and dispase-based tissue digestion protocols.

Dissociating murine skin into a single cell suspension is essential for downstream cellular analysis such as the characterization of infiltrating immune ...

Purification of Viral DNA for the Identification of Associated Viral and Cellular Proteins

The goal of this protocol is to isolate herpes simplex virus type 1 (HSV-1) DNA from infected cells for the identification of associated viral and cellular proteins by mass spectrometry. Although proteins that interact with viral genomes play major roles in determining the outcome of infection, a comprehensive analysis of viral genome associated proteins was not previously feasible. Here we demonstrate a method that enables the direct purification of HSV-1 genomes from infected cells. Replicating viral DNA is selectively labeled with modified nucleotides that contain an alkyne functional group. Labeled DNA is then specifically and irreversibly tagged via the covalent attachment of biotin azide via a copper(I)-catalyzed azide-alkyne cycloaddition or click reaction. Biotin-tagged DNA is purified on streptavidin-coated beads and associated proteins are eluted and identified by mass spectrometry. This method enables the selective targeting and isolation of HSV-1 replication forks or whole genomes from complex biological environments. Furthermore, adaptation of this approach will allow for the investigation of various aspects of herpesviral infection, as well as the examination of the genomes of other DNA viruses.

The goal of this protocol is to specifically tag and selectively isolate viral DNA from infected cells for the characterization of viral genome associated proteins.