Introduction

Genomic DNA or cDNA clones are analyzed in a simple procedure that begins with the restriction digestion of phage DNA mini preparations. The digested DNA is then assessed by agarose gel electrophoresis. For the rapid analysis, one can purify bacteriophage DNA from plate lysates as well as liquid cultures. This method explains the purification of phage DNA from plate lysates. These DNA mini preparations can be used as substrates for restriction endonucleases or as templates for PCR or RNA synthesis with the help of ‘DNA-dependant RNA polymerases’ encoded by T6, T7, and SP6 phages.

The smaller size cDNA/genomic DNA clones can then be analyzed by several methods stated below:

  • λZAP, λZipLox, and other such vectors allow recovering the inserts as plasmids which are then analyzed. This method is best suitable for smaller size inserts (less than or equal to 8kb) and beneficial for recovering cDNA from recombinant phages.
  • Specifically designed thermostable DNA polymerases that catalyze long PCRs can also be used to recover smaller inserts.
  • The insert can also be amplified using PCR to analyze cDNA clones. Using well-designed oligonucleotide primers, DNA is subjected to restriction digestion and then ligated into a plasmid. The DNA can also be directly inserted into a vector without restriction digestion. 

However, three strategies are considered for longer size clones:

  • Use a version of long-chain PCR presented by Bames in 1994 to amplify the insert before sub-cloning or analysis.
  • Rescue the insert as phagemid DNA. A phage vector with an integrated phagemid genome is used to construct the original library, which is a prerequisite in this method. 
  • Digest the recombinant DNA with restriction endonucleases. This digestion will release foreign DNA from the arms. Now, subclone this DNA directly into the plasmid vector. 

The restriction digestion product does not require any purification as vector arm to plasmid ligation products are poor bacterial transformation candidates. An equimolar plasmid to insert molar ratio is desired for establishing ligation reactions. Use a high-frequency transformation procedure to introduce foreign DNA into E. Coli. 

The larger size inserts (15kb to 20kb in size) should be screened by hybridization as the empty colonies cause high background noise. 

Once the cDNA or genomic DNA is cloned into a plasmid vector having a high copy number, we can prepare large quantities of DNA for exon hunting, restriction mapping, and DNA sequence analysis.      

By following this method, we can purify approximately 5ꭒg DNA from 5×1010 infectious particles. With the innovations in molecular biology, researchers have come up with procedures that can yield up to 23ml of 1011 PFUs. Although these procedures are lengthy and may take from 16 to 21 days, they can play a significant role in personalized phage therapy (Luong et al., 2020). 

Principle

While purifying DNA from plate lysates, the infected culture mixed with top agarose is spread over an agar plate’s surface. After incubation, the culture is eluted with TM and transferred to a microfuge tube. Cell debris is removed, and the pellet is mixed with an anion exchange resin which separates the DNA in the form of a pellet. The pellet is resuspended in TM and centrifuged again. This centrifugation is followed by phenol: chloroform extraction, and later on, DNA purification column chromatography is performed to remove unwanted contaminants. The purified DNA is resuspended in TE and stored for future analysis.

Materials

Buffers, Media, and Solutions

  • Ethanol 
  • Chloroform
  • Phenol: Chloroform (1:1 v/v)
  • TM
  • SM
  • TE (pH 8.0)
  • High Salt Buffer
    • 1M NaCl (pH 8.0)
    • 1mM EDTA 
    • 20mM Tris-Cl (pH 7.4)
  • Low Salt Buffer
    • 0.2M NaCl 
    • 20mM Tris-Cl (pH 7.4)
    • 1mM EDTA (pH 8.0)
  • LB/ NZCYM agarose plates
    • Equilibrated to room temperature
    • Agarose is preferred over agar to minimize contamination. The contaminants (inhibitors) can interrupt the enzymatic analysis of the recombinants.
  • LB/ NZCYM top agarose

Bacterial and Viral Strains

  • E. coli plating bacteria
  • Well-isolated plaques of recombinant bacteriophage λ

Other Equipment

Methods

  1. From a plate prepared from genetically pure bacteriophage stock, pick 8 to 10 well-isolated plaques with a Pasteur pipette. Mix these plaques with 1ml of SM and a drop of chloroform. 
  2. Incubate this phage suspension at 4oC for about 5 hours. This incubation facilitates the diffusion of phage particles from top agarose. 
  3. Take a sterilized culture tube and add 1 to 2 drops of bacteriophage suspension in it. Also, add 150ꭒl of plating bacteria to the tube. Place this infected culture at 37oC for 20 minutes.
  4. Melt the top agarose at 47oC and add 7ml of it to the infected culture. 
  5. Now, spread this suspension on the surface of a freshly poured NZCYM agarose plate.
  6. Incubate the plates in an inverted position at 37oC for 7-8 hours until plaques cover the plate’s entire surface. 
  7. Directly pour 7ml of TM on the surface of the top agarose. Incubate at 4oC for 4 hours with constant gentle shaking to facilitate the elution of phage particles. 
  8. Transfer the eluted phage suspension to a microfuge tube. Centrifuge the tube at 58,000rpm for 10 minutes at 4oC and discard the pellet (containing cell debris). Add a small amount of chloroform to the lysate and store the stock at 4oC. You can also remove a small aliquot of clean lysate and store it as bacteriophage stock at this step.  
  9. Take a centrifuge tube and dispense 10ml (2:1) slurry of DE52 resin in it. Centrifuge this tube at 2000rpm for 5 minutes to allow the resin to sediment. Discard the supernatant and store the resin pellet on ice.
  10. Resuspend the pellet in clear TM. Rock the centrifuge tube for 3 minutes at room temperature to let the bacteriophage particles get absorbed in the resin. 
  11. Now, centrifuge the resultant slurry for 5 minutes at 5800rpm. Transfer the supernatant to a fresh tube and repeat the centrifugation step. Remove the pellet after each centrifugation. 
  12. After the second centrifugation, transfer the supernatant to a fresh tube. Perform phenol: chloroform extraction once with the supernatant containing bacteriophage λ particles.  
  13. Shift the aqueous phase containing phage DNA to a fresh tube and add an equal volume of isopropanol. Store this mixture at -70oC for 10 minutes. 
  14. Centrifuge this mixture at 12000rpm at 4oC for 20 minutes to precipitate phage DNA. Collect the precipitated DNA. 
  15. Drain isopropanol and let the DNA pellet dry in the air. 
  16. Now, dissolve the pellet in 2ml of the low salt buffer. 
  17. Perform DNA purification column chromatography for bacteriophage purification by following the steps given below:
      1. Push 1-2ml of high-salt buffer and then 5ml of low-salt buffer with a syringe through Elutip-d (DNA purification) column.
      2. Now, attach 0.45ꭒm pre-filter to the chromatography column and push the DNA sample through it gradually. 
      3. Wash the column with 2.5ml of the low-salt buffer. 
      4. Take away the pre-filter and push 0.4ml of high-salt buffer through the column to elute DNA. 
      5. Collect the “eluate” in a microcentrifuge tube.
  18. Add 1ml of ethanol to the eluate and invert the tube multiple times. Store this mixture on ice for 20 minutes. 
  19. Centrifuge this mixture. Discard the supernatant and then wash the pellet containing DNA with 0.5ml of 70% ethanol. Now, discard the supernatant and air dry the pellet to remove the remaining ethanol. 
  20. Redissolve the pellet in a drop (50ꭒl) of TE. Dissolve the DNA by gentle tapping on the sides of the tubes and avoid vortexing. If the DNA still does not dissolve, incubate the tube at 50oC for another 15 minutes. Perform agarose gel electrophoresis to measure the quantity of phage DNA. In case the gel electrophoresis is followed by restriction digestion, digest 5-10ꭒl of the redissolved DNA. Sometimes, the DNA is resistant to restriction digestion. In this case, an additional protocol is followed.

Summary

  • cDNA or genomic DNA can be purified for further analysis either from plate lysates or liquid cultures. 
  • Sometimes, restriction digestion is performed before analyzing the DNA. The DNA is interpreted by simple agarose gel electrophoresis.
  • Different strategies are adopted for amplifying smaller and larger size clones. Smaller size clones can be amplified by using vectors like λZAP and λZipLox. PCR can also amplify small cDNA clones.
  • Larger size clones can be amplified by performing long-chain PCR, using phagemid vectors, or subcloning restrictions digested foreign DNA with plasmid vectors. 
  • Approximately 5ꭒg of DNA can be purified from 5×1010 infectious particles using the plate lysate method.

References

  1. Luong, T., Salabarria, A. C., Edwards, R. A., & Roach, D. R. (2020). Standardized bacteriophage purification for personalized phage therapy. Nature Protocols15(9), 2867-2890.
  2. Sambrook, J., & Russell, D. W. (2006). Rapid Analysis of Bacteriophage λ Isolates: Purification of λ DNA from Plate Lysates. Cold Spring Harbor Protocols2006(1), PDB-prot3984.