Hybridization based Screening of Bacteriophage DNA on the filters

Filters carrying immobilized bacteriophage DNA are labeled with 32P-labelled probes to screen by hybridization in situ. In this procedure, the filters are first submerged in a prehybridization solution (containing blocking solution) to reduce non-specific probe absorption. The filters are then incubated with a denatured radioactive probe for hybridization. Wash the filters to remove the non-specifically attached probe. Get the image of hybridization positives on an X-ray film, align the image with the plate and recover the desired hybridization positive plaques by successive screening rounds. 

The hybridization technique is also used for screening high-density gridded microarrays of a whole genomic library by using one or more than one different type of probe. The most frequently used probes in the current era include amplified PCR products, oligonucleotides, and sub-cloned DNA fragments (Campbell and Choy, 2002).  

The probes used for bacteriophage lambda screening must be pure. They should not contain any sequences that can potentially bind to the vector DNA or, in other words, are complementary to the vector. For instance, λgt11, λORFB, Charon bacteriophages, and several other bacteriophage vectors contain E. coli LacZ gene for immunochemical screening or as a part of the plasmid to rescue cloned sequences in bacteriophage λ. DNA probes are often prepared from plasmid fragments having an entire or a part of the LacZ gene. Therefore, a small amount of contamination can result in the binding of probes to the vector genome. To resolve this issue, 

1. Purify the DNA before radiolabelling by passing through two gels successively, or
2. Prepare the probe by PCR amplification and purification. 

With the advancements in molecular biology, several variations of this technique have been established. These variations include pulse-field gel Southern blot, hybridization fingerprinting, and 2D overgo hybridization. Now, the trend is shifting towards this procedure’s automation (Campbell and Choy, 2002).

Materials

Buffers and Solutions

• SM
• Wash Solution 1
  • 20x SSC (diluted to 2X) 
  • 0.1% w/v SDS
• Wash Solution 2
  • 20x SSC (diluted to 1X)
  • 0.1% w/v SDS
• Wash Solution 3
  • 20x SSC (diluted to 1X) 
  • 0.1% w/v SDS
• 2x SSPE

Nucleic Acids and Oligonucleotides

• Probe (Radiolabeled)
• Filters with immobilized bacteriophage DNA

Other Equipment

• Glass baking dish/hybridization chamber. Some investigators preferably use heat-sealable plastic bags for prehybridization and hybridization steps. These bags are opened and resealed while changing buffers. They present the following advantages:

• • It does not cause problems with hybridization.
  • These bags can be easily immersed in a water bath to ensure correct temperatures during washing and hybridization steps.

• Water bath (boiling, for denaturation of the probe). Chemiluminescent marker (You can use this marker multiple times after exposure to florescent light before every round of autoradiography)
  • Radioactive ink can be used as an alternative. To prepare it, mix a small amount of 32P with black ink. It is easier to prepare ink in three grades: very hot (>2000cps on a hand-held monitor), hot (>500cps on a hand-held monitor), and cool (>50cps on a hand-held monitor). Use a fiber tip pen to apply ink. Attach a radioactive label to the pen and store it in a safe place.

• Water-soluble glue stick 
• Whatman 3MM Paper
• Incubator (preset to hybridization temperature)

Methods

1. Thoroughly wet the dry filters from the underside by floating them on the surface of 2x SSPE. Now submerge the solution in the same for 5 minutes. Note that the color of the filter paper will change from white to bluish as the SSPE penetrates through the filter pores. Ensure that no white spots remain on the membrane before proceeding to the next step. 
2. Take prehybridization solution (3ml per 82-mm filter) in a glass dish and transfer the filters to this dish. Store the filters on a rocking platform for 60 to 120 minutes at a suitable temperature with gentle agitation to ensure that the filters are constantly washed and evenly coated in the prehybridization solution. No matter what type of container is used, ensure that filters are always fully immersed in the solution. During prehybridization, the single or double-stranded DNA bound non-specifically to the filter sites gets replaced by proteins in the blocking solution. The appropriate temperature for hybridization in 50% formamide is 42oC, and that for hybridization in an aqueous solution is 68oC. At the given temperature, formamide is less harsh on a nitrocellulose filter than an aqueous solution. However, the rate of hybridization in formamide is two to three-fold slower. Despite these advantages and disadvantages, choosing the solution is a matter of personal choice.  Nylon filters are resistant to the harmful effects of hybridization in an aqueous solution.
3. Heat 32P-labelled double-stranded probes at 100oC for 5 minutes for denaturation and then cool rapidly on ice. Do not denature single-stranded probes. Otherwise, take 0.1 volume of 3N NaOH to the probe. Transfer the probe to ice water after five minutes at room temperature and add 0.1 volume of Tris-Cl (1M and pH 7.2) and 0.1 volume of 2.5N HCl. After denaturation, store the probes at a low temperature as the DNA reannealing rate is very slow at low temperatures. Therefore, the probes remain in single-stranded form. Use 2×105 and 1×106 CPM of the probe per ml of hybridization solution. Using excess probes causes background noise (increase in non-specific hybridization), and fewer probes lower the hybridization rate. The specific activity of the 32P-labelled probe is 5x107cpm/ꭒg.
4. Add a denatured probe to the filters immersed in prehybridization solution and incubate this assembly for 12 to 16 hours at an appropriate temperature (a temperature 20-25oC below the melting point). Carry out hybridization in high ionic strength solutions (6x SSC or SSPE) to maximize the rate of binding with the probe. 6x SSPE has a high buffering capacity in the presence of formamide in the hybridization solution, so its use is favored; however, both SSPE and SSC work well. 
5. Once the hybridization is complete, quickly remove the filters from the solution and submerge them in 300-500ml of wash Solution 1 at room temperature for 5 minutes. Gently shake the filter papers and turn over the filter papers at least once. Now shift the filters to a fresh batch of washing solution and shake moderately. Repeat the step at least twice or more.  Only a small fraction of probes bind to the sequences bound to the filters. The hybridization solution can be restored to be used again. However, recycled hybridization solution yields a weaker signal because of the following possible reasons:
   1. Radioactive decay reduces the specific activity of the probe. 
   2. Probe degradation during incubation
   3. Renaturation of the denatured probe
6.  Wash the filters twice in excess of Wash Solution 2 (300-500ml) at 68oC for 60 to 90 minutes. If you are an expert, use a hand-held monitor to test whether the washing is complete. If there is still background noise, washing needs to be done at high stringencies. Dip the filters in 300-500ml of washing solution 3 at 68oC for 60 minutes. 
7. Shift the filters to a dry sheet of Whatman 3MM paper or a stack of paper towels at room temperature and let them dry. Apply glue stick beneath the filter papers and paste them on a Whatman 3MM sheet such that they do not move. Apply adhesive labels marked with radioactive ink/ chemiluminescent marker to multiple random locations on the 3MM sheet. This ink acts like a marker that aligns the autoradiograph with the filter papers. Use a Saran wrap to cover the filters along with the labels. Secure the cling film on the back of the Whatman 3MM sheet using scotch tape and then stretch the film to get rid of wrinkles. 
8. Provide X-ray exposure to the filters with an intensifying screen at -70oC for 12 to 16 hours. 
9. Now, develop the X-ray film and line it up with the filter papers with fluorescent markers or the marks left by radioactive ink. Mark the points of asymmetrically positioned dots on labeled filters using a simple non-radioactive pen (preferably red).
10. Detect the positive plaques by matching them with the plaques on the agar plate.  While preparing for stripping, place the 3MM sheet with attached filters in a tub of 2x SSC to remove the glued filter papers. The glue dissolves in this solution, and filter papers drawn from the sheet are ready to be exposed to stripping solution and another round of hybridization if required.
11. Pick a positive plaque and mix it in 1ml SM and 50ꭒl chloroform. Store all the plaques by repeating the same procedure for each of them.
12. Prepare a 50ꭒl aliquot of 10-2 dilution of the bacteriophages recovered from cored agar plug. Plate this aliquot to purify a plaque positive for hybridization. Proceed with successive rounds of screening by hybridization. A100mm plate should have approximately 300 plaques in the second round of plating. Then subject these plaques to the second round of hybridization. Now, pick a single plaque from the second round of screening and pass it through successive rounds of screening until the stock is pure and each plaque binds to its probe of interest. A recombinant isolated from the final round of screening can be used to prepare bacteriophage stocks.

Precautions

• Hybridize duplicate sets of filters to the same probe to minimize the chance of confusing positive plaque with background smudge. 

• Pick those plaques for future analysis, which yield considerable hybridization signals for both sets of filters.  

• If the alignment of filters with the plate does not permit identifying a positive plaque, core an agar plug containing many plaques in the area of interest using a sterilized Pasteur pipette. Use a fresh sterile pipette for each positive hybridization area. 

• If using heat-sealable plastic bags instead of glass dishes, secure the bag containing radioactive material inside a second non-contaminated bag.

Strengths and Limitations

This technique of probe-DNA hybridization is robust and highly sensitive. It is very specific and can identify a single recombinant from thousands of plaques. For instance, while screening the genome library for a single copy gene, you can find a single clone from 105 plaques screened. However, while screening for cDNA libraries, the number of positives depends on the abundance of the desired mRNA. 

PPCR-based screening techniques have also been established. These methods surpass the traditional hybridization-based screening in several ways, such as eliminating the signal-to-noise ratio, eliminating the need to purify phage DNA, and saving time (LeBlanc-Straceski, et al., 2006). Yet, hybridization-based screening is the most widespread method for the identification of recombinant phages.

Applications

The plaque hybridization technique has a wide range of applications in molecular biology. One such application is immunoscreening. For example, March et al. (2006) developed a new method for testing genetic vaccines. A whole genomic library was cloned into λ ZAP Express vector, which contains both prokaryotic and eukaryotic promotors upstream of the insertion site. The E. coli cells infected with phage library are immunoblotted and probed with an antiserum to recognize the candidate vaccines. These plaques are then purified and recovered as liquid lysates. The bacteriophage particles in the form of liquid lysates are directly injected into the hosts for immunization. This method was used to make a vaccine against Mycoplasma mycoides, a bovine pathogen (March et al., 2006).

Summary

• The immobilized phage DNA on the filters is hybridized with a radiolabeled probe. 
• The filters are dipped into a prehybridization solution to reduce non-specific binding, the probe is denatured, and hybridization is allowed to occur. After hybridization, filters are washed. The image of the filters is obtained on an X-ray film, and this film is then aligned with the plate. Hybridization positive plaques are purified and stored. 
• This hybridization technique is robust, highly sensitive, and specific.
• A single recombinant can be identified from thousands of plaques by using this method.

References

1. Campbell, T. N., & Choy, F. Y. (2002). Approaches to library screening. Journal of Molecular Microbiology and Biotechnology, 4(6), 551-554.
2. LeBlanc-Straceski, J., Sobrado, P., Betz, S., Zerfas, J., & Morgan, K. (2006). The lift pool method for isolation of cDNA clones from lambda phage libraries. Electronic Journal of Biotechnology, 9(4), 0-0.
3. March, J. B., Jepson, C. D., Clark, J. R., Totsika, M., & Calcutt, M. J. (2006). Phage library screening for the rapid identification and in vivo testing of candidate genes for a DNA vaccine against Mycoplasma mycoides subsp. mycoides small colony biotype. Infection and immunity, 74(1), 167-174.
4. Sambrook, J., & Russell, D. W. (2006). Hybridization of bacteriophage DNA on filters. Cold Spring Harbor Protocols, 2006(1), pdb-prot3983.