Tag: Molecular biology

As someone who has done a lot of bacteriophage research, I know how common it is to get host DNA contamination when attempting to isolate your phage genome. This issue may only become apparent after you have completed sequence analysis (nanodrop just measures any DNA, and won’t tell you the contamination). Given the high cost of sequencing in some parts of the world, the revelation can be heartbreaking. Some kits offer simple and ready-made standardised methods for a fee (significant to some of us). As someone who has worked in phage research in developing countries (Africa), I thought it would be prudent to investigate this matter further in order to find a low-cost option, as it is possible that someone else is struggling to obtain this same information.

Bacteriophages’ genetic material is either RNA or DNA, which can be circular or linear, single- or double-stranded. The isolation of phage DNA is similar to that of other microbes, though contamination from bacterial genetic materials is possible. Because the isolation requires a large number of phages (measured in PFU), stock multiplication may be necessary. The enrichment process multiplies bacteriophages. The overnight (depending on the host) bacteria culture is mixed with the phages in this approach, which is where the risk comes in. This allows any lytic phages present in the sample to infect and multiply in the target bacterial cultures.

Filtration to remove bacterial cell debris

Following the multiplication, the bacteria and other debris are removed via filtration. Filters with pore diameters of 0.22 and 0.45 microns are commonly used in this process, and they can be syringe adaptor filters or filter paper used in conjunction with the chamber and a suction pump. The latter is mostly used when the sample volume is significantly huge as in syringe adapter filters your palm is a source of required pressure. Filtration is widely used to ensure no bacteria cells pass through the filter, separating phages (filtrate) from bacteria (residue). However, because DNA molecules are small enough to pass through the pores, this does not guarantee that the bacteria’s genetic content will be stopped.

Have you ever wondered why environmental DNAse does not degrade bacterial DNA? Oh! Yes, I had the same thought, only to discover that more data demonstrating DNA survival in harsh environments had already been published. One possibility is that bacteria “absorb” free-floating foreign Genes (we have all heard of this right). This means that the enzyme did not destroy a specific molecule. As a result, my own conclusion is that DNAse will not destroy genetic material in the environment at all times and in all places, particularly on your tubes, where it may not even exist. As a matter of fact, you should introduce your own (commercially available) and be certain.

The modified phenol-chloroform-isoamyl alcohol method

The modified phenol-chloroform-isoamyl alcohol method, as used by Nale et al., (2015) is the technique I thought would be useful here. Well! It may not be as cheap as the boiling method, but it produces the best results and may be significantly less expensive than other commercial kits. In molecular biology, chloroform extraction is a liquid-liquid extraction technique used to purify nucleic acids while removing proteins and lipids. Both phenol and chloroform are non-polar solvents. Water, on the other hand, is a very polar solvent. These liquid properties are the fundamental principles of extracting DNA using this method (we won’t go deep into that). But let’s dive into the procedure itself.

The procedure of extracting phage genomic DNA using the modified phenol-chloroform-isoamyl alcohol method

1. Multiply your phage by the titer of 10⁹ PFU/ml
2. Use a clean and sterile pipette tip to pick 1 ml of bulked phage lysate and dispense it into a clean falcon tube.
3. Add 12.5 mM MgCl₂, 0.8 U/ml of DNase, and 0.1 mg/ml of RNase (final concentrations) to the falcon tube and incubated at room temperature for 1 h to eliminate bacterial DNA.
4. Add 20 mM of EDTA, 0.5 mg/ml of proteinase K, and 0.5% of sodium dodecyl sulfate, before incubation at 55°C for an additional hour to digest the phage capsid. EDTA is a chelating agent, it removes the Mg ion, rendering the DNase inactive due to a lack of this divalent cation. Without this DNAse, your phage DNA will undoubtedly be digested.
5. Mix an equal volume of phenol-chloroform-isoamyl alcohol (25:24:1, vol/vol/vol) and centrifuge at 15,000 × g for 5 min to extract the DNA.
6. Treat the aqueous layer obtained with 0.3 M sodium acetate and 2 volumes of ice-cold ethanol to precipitate the DNA.
7. After incubation for 10 min on ice, elute the DNA by centrifugation at 21,000 × g for 20 min.
8. Wash the resultant pellet with 0.5 ml of 70% ethanol.
9. Dissolve the pellet in 5 mM Tris HCl. Boom! There you have your phage DNA
10. Quantify and analyze the DNA (Nanodrop things……………..)

For many other protocols click Here

References

1. Nale J. Y., Spencer J., Hargreaves K. R., Buckley A. M., Trzepinski P., Douce G. R., et al. (2015). Bacteriophage combinations significantly reduce clostridium difficile growth in vitro and proliferation in vivo. Antimicrob. Agents Chemother. 60 968–981. 10.1128/AAC.01774-15.
2. Grami, E., Badawy, S., Kiljunen, S., Saidi, N., & Skurnik, M. (2023). Characterization and genome analysis of Escherichia phage fBC-Eco01, isolated from wastewater in Tunisia. Archives of Virology, 168(2), 1-10.
3. Ji, Y., Xi, H., Zhao, Z., Jiang, Q., Chen, C., Wang, X., … & Gu, J. (2023). Metagenomics analysis reveals potential pathways and drivers of piglet gut phage-mediated transfer of ARGs. Science of The Total Environment, 859, 160304.
4. Chernyshov, S. V., Tsvetkova, D. V., & Mikoulinskaia, G. V. (2023). A rapid and efficient technique for the isolation of Bacillus genomic DNA using a cocktail of peptidoglycan hydrolases of different type. World Journal of Microbiology and Biotechnology, 39(1), 1-10.