1Department of Microbial and Environmental Genomics, The J. Craig Venter Institute, 2Department of Synthetic Biology and Bioenergy, The J. Craig Venter Institute
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Fadrosh, D. W., Andrews-Pfannkoch, C., Williamson, S. J. Separation of Single-stranded DNA, Double-stranded DNA and RNA from an Environmental Viral Community Using Hydroxyapatite Chromatography. J. Vis. Exp. (55), e3146, doi:10.3791/3146 (2011).
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’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.
1. Preparation of Solutions
Before performing hydroxyapatite chromatography, phosphate buffers must be prepared and the hydroxyapatite must be properly hydrated.
2. Preparation of Econo-Column
3. Hydroxypaptite Chromotography
4. Desalting Nucleic Acid Samples
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 “community” 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.
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°C and applied to a hydroxyapatite column maintained at a constant 60°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.
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.
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.
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 is important to test each preparation using known nucleic acids (e.g. from a cultivated virus preparation). This will allow the user to ascertain the specific concentrations of phosphate-containing buffers needed to elute the desired nucleic acids.
A limiting factor of this technique is the quantity of available Ca2+ ions present in the hydroxyapatite that can bind the phosphate backbone of the nucleic acids. The capacityamount of the hydroxyapatite in the column can be scaled up or down to column (dictated by the size of water-jacketed column and amount of hydroxyapatite used) and the volume of buffers used for elution can be scaled up or down to accommodate very small or very large quantities of input nucleic acids, but is ultimately limited by the size of the water-jacketed column.
Additionally, a batch technique where the nucleic acids, hydroxyapatite, and phosphate-containing buffers are combined in a tube, mixed, and centrifuged at low speed is an alternative way of isolating targeted nucleic acids. This strategy may be beneficial in some instances, but is not as reliable as the chromatographic methods which are more accurate and provide better fractionation of nucleic acids.
No conflicts of interest declared.
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’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.
|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|
|10% SDS Solution||Invitrogen||24730-020||UltraPure, 1L|
|0.5M EDTA||Invitrogen||AM9262||pH 8.0, 1L|
|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|