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Southern Blot

Written by Super User. Posted in DNA

Learn about southern blot.

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Background on Southern Blotting

History of the Southern Blot Technique

Southern blotting is a technique named after its inventor and developer, the British biologist Edwin M. Southern in 1975. Southern blotting is a technique which allows the detection of a specific DNA sequence (gene or other) in a large, complex sample of DNA (e.g. cellular DNA).

"Before PCR and cheap fast sequencing changed our view of the universe that is genetics, the Southern Blot was a universal workhorse. There was not an experiment in molecular genetics which did not at some stage employ a Southern Blot. It is still a useful tool and you need to know about it so that you can interpret historical data."

Southern Blotting Procedure

southern blot

Diagram of the Southern blot technique.Figure is Copyright. If you would like to use it for a lecture or science project, please contact us for permission.

In Step 1, DNA is digested with restriction endonucleases. High-molecular-weight DNA is digested into smaller fragments.

In Step 2, The DNA is then separated out by size by electrophoresis on an agarose gel.

In Step 3, a sheet of nylon or nitrocellulose membrane (purple) is placed on top of the agarose gel in a buffer solution. This is considered the blotting or transferring stage. Pressure is applied evenly to the gel using either suction or by placing a stack of paper towels and a weight on top of the membrane and gel to ensure even contact between gel and membrane. Buffer transfer by capillary action from a region of high water potential to a region of low water potential (usually filter paper and paper tissues) is then used to move the DNA from the gel on to the membrane. Positively charged membrane (purple) is usually used which allows the DNA to bind with high affinity to the membrane through ion interactions between the negatively charged DNA and positively charged membrane.

In Step 4, nitrocellulose membranes are baked by exposure to high temperatures (from 60 to 100 °C). Nylon membranes are exposed to UV radiation. These steps are used to ensure the permanent and covalent crosslink of the DNA present in the bands to the membranes.

In Step 5, the membrane is exposed to a radiolabeled probe. This probe is a single-stranded DNA fragment which has your sequence of interest that you want to detect. This probe is incubated with the membrane and allowed to hybridize with DNA on the membrane. Probes are usually radiolabeled so that they may be detected on film, however newer probes are also used which are non-radioactive such as fluorescent or chromogenic dyes. After hybridization, excess unbound probe is washed away from the membrane, leaving specifically bound probe.

In Step 6, the pattern of hybridization is detected by visualization on X-ray film by autoradiography in the case of a radioactive or fluorescent probes, or by development of color on the membrane if a chromogenic detection method is utilized.

(Notes for Step 3: If large DNA fragments are present greater than 15 kb in size may be depurinated by then dilute HCl acid prior to blotting which will break the DNA into smaller pieces, thus allowing more efficient transfer from the gel to membrane. If alkaline transfer methods are used, the DNA gel is placed into an alkaline solution (typically containing sodium hydroxide) to denature the double-stranded DNA. The denaturation in an alkaline environment provides for improved binding of the negatively charged DNA to a positively charged membrane, separates it into single DNA strands for later hybridization to the probe (see below), and destroys any residual RNA that may still be present in the DNA.

 

Overview

Molecular Biology AudioAUDIO: Listen to a detailed Southern Blot Explanation! Courtesy: National Human Genome Research Institute.

As mentioned, the Southern blot is a technique used to identify and locate DNA sequences which are complementary to another piece of DNA called a probe.

The separation on an electrophoretic gel of sequences or fragments of genomic or complementary DNA, partially digested by endonucleases, The fragments are then 'blotted' onto a membrane and allowed to hybridize with a specific, labeled probe in order to detect which bands contain the fragment or gene of interest. Southern blot is particularly useful in detecting large gene rearrangements/deletions and large trinucleotide repeat expansions.

A Southern blot is a method routinely used in molecular biology to check for the presence of a DNA sequence in a DNA sample. Southern blotting combines agarose gel electrophoresis for size separation of DNA with methods to transfer the size-separated DNA to a filter membrane for probe hybridization. Other blotting methods (i.e., western blot, northern blot, southwestern blot) that employ similar principles, but using RNA or protein, have later been named in reference to Southern's name. As the technique was eponymously named, Southern blot should be capitalised, whereas northern and western blots should not.

It is also used to determine the molecular weight of a restriction fragment and to measure relative amounts in different samples.

Blots are techniques for transferring DNA and RNA proteins onto a carrier so they can be separated, and often follows the use of a gel electrophoresis. The Southern blot is used for transferring DNA.

 

Hybridization of the probe to a specific DNA fragment on the filter membrane indicates that this fragment contains DNA sequence that is complementary to the probe.

Applications of the Southern Blot Method

Southern blots are used in several main areas including gene discovery and mapping, evolution and development studies, diagnostics and forensics.

  • Southern blots allow investigators to determine the molecular weight of a restriction fragment and to measure relative amounts in different samples.
  • Southern blot is used to detect the presence of a particular bit of DNA in a sample
  • analyze the genetic patterns which appear in a person's DNA.
  • analyze restriction digestion fragmentation of DNA or a biological sample
  • In regards to genetically modified organisms, Southern blotting is used as a definitive test to ensure that a particular section of DNA of known genetic sequence has been successfully incorporated into the genome of the host organism.

 

Detection limit of Southern Blotting

Under optimal conditions, Southern blotting detects ~ 0.1 pg of the DNA of interest

Southern Blot Buffer Recipes

20X SSC Buffer

To make 1 liter of 20X SSC Buffer, add the following:

  • 3M NaCl: 175.3 g
  • 0.15M Na Citrate: 88.2 g
  • dH2O: 800 mls
  • pH to 7.0 with HCl (0.1-0.5 mLs approx.)

Southern Blot Protocol

  1. Photograph gel with ruler.
  2. Denature the gel for 30 mins at RT (room temp), shaking, with 0.5 M NaOH, 1.5 M NaCl
  3. Rinse with H2O 2x-3x times
  4. Neutralize: 30 mins., RT, shaking
  5. While neutralizing, cut membrane to fit gel, cut 3mm paper (3-4 sheets) to size of the gel, prepare appropriate size paper towels for blotting.
  6. Set up blotting: 2 layers of 3mm paper as wicks into 20X SSC. Gel on top of wicks, remove any bubblesPre-wetted membrane (in 2xSSC) on top of gel
  7. 3mm paper on top of membrane, Paper towels on top of 3mm paper. 500g weight on top of paper towels.
  8. 6-18 hrs to blot
  9. Air dry filter, UV irradiate 3.5 min
  10. Wash filter in hot 2x SSC 0.1 % SDS twice

Troubleshooting and Problems? See the DNAse Footprinting Forum!

For problems and troubleshooting Southern Blotting, post a new thread to discuss it in the Southern Blotting Forum!

Other DNA Protocols and Methods

DNA Cloning Guide

DNA Gel Electrophoresis

Restriction Enzyme Digestion - all you need to know !

Genomic DNA Isolation Protocol

Baceteria DNA Isolation

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