Name: separation of single-stranded dna, double-stranded dna and rna from an environmental viral community using hydroxyapatite chromatography
Uploaded: 2011-09-29T13:00:00
Description: 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

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...

<ol>
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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, ...

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