The gel electrophoresis technique separates the DNA fragments based on their sizes. Smaller molecules move faster than the larger molecules. A basic question arises as to why separate these fragments? The reason is very simple. Separation of fragments based on the sizes helps to obtain specific fragments from the gel instead of getting the entire genomic DNA. Hybridization involves finding the location of a gene or its product using a nucleic acid probe. Most of the times, the probes are small single-stranded DNA molecules. Determination of complementary sequences utilizes hybridization techniques. Hence, the probes bind only to the complementary sequences. Isolated bands from electrophoretic technique determine an efficient mapping of DNA sequences or gene detection. Blotting technique facilitates hybridization. The process involves the transfer of bands to a nitrocellulose membrane. There are three types of blotting procedures depending on the type of the molecule. Southern blotting is used to blot the DNA. Northern blotting is used to blot the RNA. Western blot involves the transfer of protein bands. E.M. Southern derived the southern blotting method for the first time.
Image: Southern blotting
Analyzing the sequences using southern blotting:
Step 1: Treatment with a restriction enzyme:
The DNA undergoes a treatment with a restriction enzyme. The enzymes cleave the DNA into various fragments. The process of cleaving the DNA to obtain fragments is known as restriction digestion. The fragments obtained from restriction digestion are known as restriction digests. Restriction enzymes are known as molecular scissors as they cut the DNA at specific sites known as the restriction sites.
Step 2: Separation of the fragments through gel electrophoresis:
The main aim of restriction digestion involves studying the DNA in bits and pieces and picking up the piece of interest for analyzing. Electrophoresis does the work of separating the fragments as per the sizes. Not only DNA but also RNA can be separated. The principle of electrophoresis is simple. The DNA is a negatively charged molecule. It migrates toward the positive electrode. A positively charged molecule moves toward the negative electrode. The shape of the molecule, the charge, and the molecular length determine the rate of migration. Only one criterion of gel electrophoresis involves molecular length. The composition of the gel mainly constitutes agarose, which is nothing but a network of pores through which DNA molecules travel. Molecules of different lengths form bands on the gel.
Step 3: Staining the DNA
Staining the DNA involves ethidium bromide. This chemical is a carcinogen and neurotoxic. Use it with precaution. Staining with the ethidium bromide helps in visualizing the bands under ultraviolet light. Ethidium bromide intercalates with the DNA.
Step 4: Transferring the gel to a membrane filter:
The gel consisting of DNA fragments gets transferred to the membrane filter. Following description is about the apparatus. Firstly, a buffer solution poured into a tray serves as an alkaline medium. Soaking the gel in the buffer solution denatures the DNA into single strands. Next step involves neutralization of the gel and placing the blotting paper. The ends of the paper act as a wick that takes up the buffer solution until the gel. The membrane filter covers the gel. The next step involves placing the paper towels and weight on the filter. Due to the blotting action, the buffer solution travels through the gel onto the membrane filter. The DNA fragments get picked up by the buffer solution and get transferred to the membrane.
Step 5: Hybridization with the probes:
The probes may or may not be radioactively labeled. The process involves the addition of the probe to the membrane filter so that the DNA present on the filter gets hybridized with the probe.
Step 6: Autoradiography:
Permanent fixation of the DNA on the membrane involves heating at 800C for 2-3 hours. Now, the DNA gets completely hybridized with a labeled DNA probe. The probe forms a complementary base pair with the homologous sequence on the DNA fragment. Unbound probes are removed by carefully washing the membrane. Autoradiography technique involves an X-ray sensitive photographic film. Exposing the membrane filter to the X-ray sensitive photographic film determines the labeled molecules.
In summary, the southern blotting technique involves restriction digestion, gel electrophoresis, probing and autoradiography.
Applications of the southern blotting:
1. SNP analysis:
Single base pair changes constitute single nucleotide polymorphisms. Southern blotting efficiently determines SNP alleles. The initial step involves the isolation of genomic DNA and digestion with the restriction enzymes. The electrophoretic techniques separate the fragments based on their sizes in kilobases. The action of the blotting paper helps in transferring the DNA present on the gel to the membrane filter placed on top of the gel. A stack of paper towels and weight kept above the membrane helps in keeping the membrane fixed at one place. Hybridization with the probe enables complementary base pairing. The visualization of the bands under an X-ray sensitive photographic film gives a clear picture of the DNA. Southern blotting helps in detecting homozygotes and heterozygotes. Comparison of bands becomes easy with the southern blotting.
2. DNA molecular testing with ASOs:
It includes allele-specific oligonucleotide hybridization or short oligonucleotides complementary to SNP alleles. The oligonucleotides mixed with DNA get hybridized. ASO hybridization also involves Southern blotting. The ASOs labeled radioactively get hybridized with the DNA immobilized on the membrane filter. Analysis of the resulting autoradiograms helps in detecting gene mutations.
3. RFLP analysis:
RFLP analysis includes detection of genetic disorders such as PKU, sickle cell anemia, and many others. Restriction fragment length polymorphisms or RFLP analysis exploits homologous DNA variations.
4. Zoo blot:
A blot consisting of DNA from a variety of organisms is known as a zoo blot. The digestion of DNA obtained from organisms such as chicken or a hamster with the restriction enzymes gives fragments of different lengths. The analysis of these fragments includes southern blotting.
5. DNA typing or DNA fingerprinting:
Digestion of DNA with endonucleases giving fragments, later on electrophoresed, give banding patterns on the gel. The southern blot of the probe gets further probed with the VNTR-specific probes in the DNA fingerprinting technique. Applications of DNA fingerprinting include paternity and maternity testing, studying mitochondrial inheritance, and crime scene investigation.
6. DNA microarray involving southern blotting:
Southern hybridization with DNA microarray includes unlabelled DNA probes targeting label-free DNA molecules. However, DNA microarray uses a chip or a probe array. The method uses fluorescence dyes or cyanine dyes.
References:
[1] Molecular Biology Techniques: An Intensive Laboratory Course, Walt Ream, Katharine G. Field
References:
[1] Molecular Biology Techniques: An Intensive Laboratory Course, Walt Ream, Katharine G. Field
[2] Ana Techniques in Biotechnology, Goutam Bhowmik
[3] Gene Cloning and DNA Analysis, T.A. Brown
[3] Gene Cloning and DNA Analysis, T.A. Brown
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