Steps of Southern Blotting Technique

In this steps of southern blotting technique post we have briefly explained about southern blotting, principle, components, southern blotting procedure, applications, advantages and limitations of southern blotting procedure.

Definition

The technique of transferring DNA fragments separated by electrophoresis onto a membrane for immobilisation and identification is known as a southern blotting procedure.

One of the most important procedures in molecular biology is southern blotting procedure. Southern blotting is a technique that involves transferring DNA molecules, mainly restriction fragments, from an electrophoresis gel to a nitrocellulose or nylon sheet in such a way that the gel’s DNA banding pattern is replicated on the membrane. It was invented by E. M. Southern in 1975.

The DNA becomes immobilised on the membrane during transfer or as a result of further treatment, and can be utilised as a substrate for hybridization analysis with labelled DNA or RNA probes that precisely target individual restriction fragments in the blotted DNA.

Southern blotting procedure is, in essence, a technique for detecting a specific restriction fragment against a background of many other restriction fragments.

The restricted DNA could be a plasmid or bacteriophage clone, in which case Southern blotting is used to confirm the identity of a cloned fragment or to identify an interesting sub-fragment from within the cloned DNA, or it could be genomic DNA, in which case Southern blotting procedure is used as a precursor to techniques like restriction fragment length polymorphism (RFLP) analysis.

Principle

The ability of nitrocellulose powder or sheets to bind DNA has been known for many years and was utilized in the 1950s and 1960s in various types of nucleic acid hybridization studies.

In these early southern blotting procedure the immobilized DNA was unfractionated, simply consisting of total DNA that was bound to nitrocellulose powder or spotted onto a nitrocellulose sheet.

The introduction in the early 1970s of gel electrophoresis methods that enable restriction fragments of DNA to be separated on the basis of their size prompted the development of techniques for the transfer of separated fragments en masse from gel to nitrocellulose support.

The procedure described by Southern (1975), involving capillary transfer of DNA from the gel to a nitrocellulose sheet placed on top of it, was simple and effective, and although embellished over the years this original procedure differs only slightly from the routine method still used in many molecular biology laboratories.

Requirements

Materials

  1. Agarose gel
  2. Cellulose acetate membrane
  3. Centrifuge Tubes
  4. Film processor
  5. Glass Plate
  6. Hybridization Bottles
  7. Hybridization Oven
  8. Nitrocellulose membrane
  9. Power supply
  10. UV radiation
  11. Water Bath
  12. Whatman 3 mm paper

Chemicals

  1. Agarose
  2. Bovine serum albumin
  3. DNA labeling kit
  4. DNA loading buffer
  5. Formamide
  6. Nucleic acid detection kit
  7. Phenol
  8. Polyvinylpyrrolidone
  9. Restriction Enzymes
  10. Restriction enzymes buffer
  11. Sodium chloride
  12. Sodium citrate
  13. Sodium dodecyl sulfate (SDS)
  14. Sodium hydroxide
  15. TBE buffer
  16. Tris base

Reagents

1. Denaturation buffer: Sodium hydroxide (NaOH) and Sodium chloride (NaCl) in the ratio 1:6

2. Neutralization buffer: Tris hydrochloride (Tris-HCl) and Sodium chloride NaCl in the ratio 5:3

3. SSC: 175.3 g of Sodium chloride (NaCl) and 88.2 g of Sodium citrate to 1L of distilled water.

4. Detection Buffer: Tris hydrochloride (Tris-HCl) and Sodium chloride (NaCl) in the ratio 5:1.

Southern Blotting Procedure

1. DNA restriction

In a 500 mL microtube, place 10 mg of deoxyribose nucleic acid. 4 mL of 10 restriction enzyme buffer is added.

By adding the distilled water, the final volume should be 40 mL. 1mL of Bcl1 restriction enzyme is added. The mixture should be vortexed, pulse-spun, and incubated at 50°C overnight.

Southern blotting

Southern Blotting Procedure Steps

2. Gel Preparation

Assemble a gel casting tray and gel comb measuring 25 cm in length. 300 mL 0.5 percent agarose (300 mL 1 Tris-borate ethylene diamine triacetic acid (TBE) and 1.5 g agarose) Heat the agarose in a microwave or on a hot plate to dissolve it.

Allow it to cool to 55–60°C before adding 25 mL ethidium bromide. Pour it into the casting tray, remove any air bubbles, and set it aside to dry.

3. Electrophoresis

Prepare the samples after incubation and add 1.5 mL of 10% sodium dodecyl sulfate (SDS) and 8 mL of 6× sucrose loading buffer.

Remove the gel comb, place in the electrophoresis tank, and cover with 1× TBE. Carefully load the samples into the gel and run the samples on the gel from negative to positive at 45 V, for overnight.

4. Denaturation

Examine the gel with the transilluminator. Make a note of the sizes of the marker’s bands to locate the area of interest.

Inject a small amount of ink into the position of two of the marker’s bands closest to the area of interest. Isolate the targeted section by cutting the top and bottom of the gel.

Transfer it to a sandwich box and depurinate it in 0.25 M HCl for 10-15 minutes on an orbital shaker.

Replace the HCl with 500 mL denaturing buffer (1.5 M NaCl, 0.5 M NaOH) and place the sandwich box on an orbital shaker at room temperature for 60 minutes at low speed.

5. Blotting

A somewhat larger oblong sponge is placed on a glass dish filled with SSC, leaving the sponge half-submerged in the buffer.

Three pieces of 3mm Whatman paper, the same size as the sponge, are cut. Wet the sponge with SSC after placing these on it.

The gel is placed on the filter paper and rolled over the surface using a glass pipette to eliminate bubbles.

On top of the gel, a nylon membrane just large enough to cover the surface is placed. SSC is then pumped into the membrane, and a few sheets of filter paper are placed on top of it. The DNA transfer is allowed to occur overnight.

6. Baking/ Immobilization

The DNA strands on the membrane can also be immobilised by exposing the membrane to UV radiation after it has been withdrawn from the blotting structure and placed in a vacuum or conventional oven at 80°C for 2-3 hours.

7. Hybridization

The hybridization probe, which can be a DNA fragment or an RNA segment with a specific sequence that recognises the target DNA, is exposed to the membrane.

By adding radioactivity or labelling the molecules with fluorescent or chromogenic dye, the probe nucleic acid can be identified.

The parameters for the process are selected so that the probe hybridises with a complementary sequence on the membrane and hybridises with the target DNA.

After hybridization, the probes that are bound nonspecifically or remain unbound are removed with a buffer, leaving only labelled probes coupled to the target sequence.

8. Detection

The hybridized regions on the membrane can be detected via autoradiography by placing the nylon membrane in contact with a photographic film.

The images indicate the position of the hybridized DNA molecules, which can be used to determine the length of the fragments by comparing them with the marker DNA molecules of known length.

Similarly, the images also provide information about the number of the hybridizing fragments and their size.

If a fluorescent or a chromogenic dye is used, these can be visualized on X-ray film or by the development of color on the membrane.

If bands are weak, replace the cassette in the freezer for 24-48 hours before the second film.

Observation

A southern blotting procedure produces bands on the membrane as a result of the results. By comparing the relative sizes of the DNA fragments to DNA bands of known lengths, the size of the DNA fragments can be estimated.

Applications

1. In the fields of gene discovery, mapping, evolution, and diagnostic research, southern blotting procedure offers a wide range of applications.

2. Southern blotting procedure can be used to detect point mutations and other structural rearrangements in DNA sequences during DNA analysis.

3. Southern blotting procedure also allows for the determination of restriction fragment molecular weights, which aids in the analysis of such fragments.

4. Because southern blotting procedure allows for the detection of a specific DNA fragment, southern blotting procedure can be utilized in fingerprinting for personal identification.

5. Southern blotting procedure can be utilized for disease diagnosis as well as genetic disease prenatal diagnosis.

Advantages and Limitations

1. Southern blotting procedure is a good way to look at lengthy sections of DNA, such multikilobase restriction fragments.

2. When compared to other genomic analysis approaches, the expression levels and profiles are more easily observed in Southern blotting procedure.

3. Southern blotting procedure has a wide range of applications in DNA fingerprinting, including paternity and maternity testing, forensic studies, and personal identification; Southern blotting procedure also less expensive than genome sequencing and can be used to quantify the amount of DNA.

4. When compared to other genome analysis approaches, the Southern blotting procedure may necessitate a substantial amount of high-quality DNA.

Further Readings

Reference

  1. https://www.alliot.fr/BIO/PDF/SouthernBlot-.pdf
  2. https://www.genome.gov/genetics-glossary/Southern-Bloth
  3. https://www.thesciencenotes.com/southern-blotting-principle-procedure-and-applications/
  4. https://conductscience.com/southern-blotting-protocol/
  5. https://www.gbiosciences.com/image/pdfs/protocol/BE-315_protocol.pdf