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

Summary

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.

Abstract

Viruses, particularly bacteriophages (phages), are the most numerous biological entities on Earth^1,2. 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’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.

Protocol

1. Preparation of Solutions

Before performing hydroxyapatite chromatography, phosphate buffers must be prepared and the hydroxyapatite must be properly hydrated.

1. 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 increa....

Discussion

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˜ 0and 0.40M respectively. However, each preparation of hydroxyapatite can have a slightly different composition and elution profile so it .......

Materials

List of materials used in this article

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