Purification of filamentous phage ssDNA genomes using Chaotropic salt/silica methods
Abstract
Filamentous phages play important roles in molecular biology and bacterial pathogenesis. There is therefore great interest in the genomes of the phages, including M13 and Pf, which infect Escherichia coli and Pseudomonas aeruginosa, respectively. However, efficient methods for purifying single-stranded DNA (ssDNA) from filamentous phages are scarce and rely mainly on repurposed commercial kits developed for dsDNA. These kits yield variable amounts of ssDNA, varying considerably from one phage to another, their solutions are proprietary and troubleshooting, and customizing the protocol to improve purification is not possible. Here, we developed and optimized two protocols based on ssDNA binding to silica columns in the presence of guanidine hydrochloride and potassium iodide chaotropic salts, for filamentous phages M13KO7 and Pf4. The protocols achieved 60 to 100% recovery, equal to or higher than the Monarch Spin gDNA Extraction and DNeasy Blood and Tissue kits, with similar A260/280 and A260/230 ratios, intact DNA in agarose gels, and no detectable host genomic DNA by PCR. Recovery depended critically on the chaotropic salts and on ethanol, and did not increase at lower pH, consistent with a water-exclusion model in which hydrophobic interactions and hydrogen bonding dominate over electrostatic repulsion. Phage-specific differences in recovery indicate that optimization may be needed for some phages, which the fully customizable nature of these protocols facilitates. Comparing the virion ssDNA with the double-stranded replicative form (RF) revealed genome variants in both phages, including a shorter M13KO7 form lacking the kanamycin resistance-p15A insert, and illustrates the value of analyzing both RF and ssDNA forms to study filamentous phage biology. Implementing these protocols in routine filamentous phage workflows should facilitate their characterization and engineering.
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BibTeX
@misc{perezrodriguez2026purification,
title = {{Purification of filamentous phage ssDNA genomes using Chaotropic salt/silica methods}},
author = {Saumel Perez-Rodriguez and Arya Khosravi and Carlos Milla and Paul L Bollyky},
howpublished = {bioRxiv},
year = {2026},
month = oct,
doi = {10.64898/2026.10.21.760820},
url = {https://www.biorxiv.org/content/10.64898/2026.10.21.760820v1},
note = {CC BY-ND},
}
RIS
TY - UNPB
TI - Purification of filamentous phage ssDNA genomes using Chaotropic salt/silica methods
AU - Saumel Perez-Rodriguez
AU - Arya Khosravi
AU - Carlos Milla
AU - Paul L Bollyky
PY - 2026
DA - 2026/10/21
PB - bioRxiv
DO - 10.64898/2026.10.21.760820
UR - https://www.biorxiv.org/content/10.64898/2026.10.21.760820v1
AB - Filamentous phages play important roles in molecular biology and bacterial pathogenesis. There is therefore great interest in the genomes of the phages, including M13 and Pf, which infect Escherichia coli and Pseudomonas aeruginosa, respectively. However, efficient methods for purifying single-stranded DNA (ssDNA) from filamentous phages are scarce and rely mainly on repurposed commercial kits developed for dsDNA. These kits yield variable amounts of ssDNA, varying considerably from one phage to another, their solutions are proprietary and troubleshooting, and customizing the protocol to improve purification is not possible. Here, we developed and optimized two protocols based on ssDNA binding to silica columns in the presence of guanidine hydrochloride and potassium iodide chaotropic salts, for filamentous phages M13KO7 and Pf4. The protocols achieved 60 to 100% recovery, equal to or higher than the Monarch Spin gDNA Extraction and DNeasy Blood and Tissue kits, with similar A260/280 and A260/230 ratios, intact DNA in agarose gels, and no detectable host genomic DNA by PCR. Recovery depended critically on the chaotropic salts and on ethanol, and did not increase at lower pH, consistent with a water-exclusion model in which hydrophobic interactions and hydrogen bonding dominate over electrostatic repulsion. Phage-specific differences in recovery indicate that optimization may be needed for some phages, which the fully customizable nature of these protocols facilitates. Comparing the virion ssDNA with the double-stranded replicative form (RF) revealed genome variants in both phages, including a shorter M13KO7 form lacking the kanamycin resistance-p15A insert, and illustrates the value of analyzing both RF and ssDNA forms to study filamentous phage biology. Implementing these protocols in routine filamentous phage workflows should facilitate their characterization and engineering.
SN - 2692-8205
ER -