The following points highlight the top seven blotting techniques used in detection of DNA. The techniques are: 1. Southern Blotting 2. Northern Blotting 3. Western Blotting 4. South-Western Blotting 5. North-Western Blotting 6. Dot Blotting 7. Zoo Blotting.
Technique # 1. Southern Blotting:
Southern blot hybridization detects target DNA fragments that have been size-fractionated by gel electrophoresis. This technique was invented in 1975 by E.M. southern.
In this technique we exploit the property of a radio-labelled probe with the single stranded DNA. If we want to detect the presence of a specific sequence in our mixed DNA sample then we will accordingly design the probe which will have complementary sequence to our target sequence.
In this procedure, called a Southern blot, DNA from the sample is cleaved into restriction fragments with a restriction endonuclease, and the fragments are spread apart by gel electrophoresis. The double-stranded helix of each DNA fragment is then denatured into single strands by making the pH of gel basic, and the gel is “blotted” with a sheet of nitrocellulose, transferring some of the DNA strands to the sheet.
Next, a probe consisting of purified, single-stranded DNA corresponding to a specific gene (or mRNA transcribed from that gene) is poured over the sheet.
Any fragment that has a nucleotide sequence complementary to the probe’s sequence will hybridize (base pair) with the probe. If the probe has been labelled with 32P, it will be radioactive, and the sheet will show a band of radioactivity where the probe is hybridized with the complementary fragment.
Information Obtained from Southern Blotting:
We can get following information from the technique of Southern blotting;
1. Whether a particular gene is present and how many copies are present in the genome of an organism.
2. The degree of similarity between the chromosomal gene and the probe sequence.
3. Whether recognition sites for particular restriction endonucleases are present in the gene. By performing the digestion with different endonucleases, or with combinations of endonucleases, it is possible to obtain a restriction map of the gene, i.e., an idea of restriction enzyme sites in and around the gene which will assist in attempts to clone the gene.
4. Whether re-arrangements have occurred during the cloning process.
Uses of Southern Blotting:
1. To identify a single gene among thousands of fragments of DNA and to detect sequences of DNA in an organism’s genome.
2. Used in gene discovery and gene mapping.
3. To analyse the genetic patterns in an organism’s DNA.
4. To identify gene mutation, deletion, duplication, and gene rearrangement involved in diseases.
5. To determine the number of copies of a particular DNA sequence presented in the genome of an organism.
6. To identify related DNA sequence in the genome and to determine if there is a gene family (a group of similar genes).
7. To detect certain cancers and genetic diseases, such as:
a. Monoclonal leukemia population
b. Sickle cell mutation.
8. Used in DNA fingerprinting, genetic engineering and forensic science for tests as under:
a. Paternity testing
b. Personal identification
c. Sex determination
d. Species exclusion.
Technique # 2. Northern Blotting:
Northern blotting is a simple extension of Southern blotting, and derives its name from the earlier technique. It is one of the key techniques in molecular biology, its principal aim being the measurement of RNA (in particular mRNA).
RNA molecules are separated by size and detected on a membrane using a hybridization probe with a base sequence complementary to all, or a part, of the sequence of target RNA.
RNA is extracted from the cells of interest, but precautions must be taken to avoid degradation of single-stranded RNA by ribonuclease (RNase), which is found on the skin and on glassware. Wear gloves use specially treated plastics and glassware to avoid accidental introducing ribonuclease to extraction prep.
Addition of di-ethyl-pyro-carbonate (DEPC) inhibits ribonuclease activity and also baking at high temperature destroys ribonuclease activity (only useful for treating heat resistant equipment like glassware).
It is performed in following steps:
RNA is isolated from several biological samples (e.g., various tissues, various developmental stages of same tissue, etc.)
The RNA samples are separated according to their size on an agarose gel.
The gel is then blotted on a nylon membrane or a nitrocellulose filter.
The membrane is placed in a dish containing hybridization buffer with a labelled probe. RNA blots are most usually probed with cDNA fragments.
The membrane is washed to remove unbound probe.
The labelled probe is detected via autoradiography (if a radioactive probe is used) or via a chemiluminescence reaction (if a chemically labelled probe is used). In both cases this results in the formation of a dark band on an X-ray film.
Information Obtained from Northern Blotting:
We can get following information from the technique of Southern blotting;
1. Differential expression patterns of a particular gene.
(a) In which tissues it is expressed.
(b) If it is expressed during certain stages of development.
(c) If expression changes under different conditions/treatments of the cell.
3. Whether a genomic DNA probe has regions that are transcribed.
Uses of Northern Blotting:
1. Northern blotting allows researchers to determine gene expression patterns. This indicates a myriad of practical applications, allowing researchers to compare patterns of gene expression in cells of tissues, cells of patients undergoing treatment and cells of different developmental stages.
2. Northern blot analysis can also be used to detect cancerous pancreatic cells and tissues. In one study the researchers review that the pancreatic cancers exhibited 3- fold, 10-fold and 15-fold increase in mRNA of a certain receptor, indicating for the first time that this receptor was involved in carcinogenesis of pancreatic cancer. This information has been obtained by using Northern blotting technique.
3. Northern blotting can also enable the scientists to know the function of unknown proteins.
4. This technique enables the scientists to detect the size of RNA.
5. It also allows them to observe the alternate splice products, using the probes with partial homology.
Technique # 3. Western Blotting:
The Western Blot is an analytical technique used to detect specific proteins in a given sample of tissue homogenate or extract. Sometime referred to as immune blotting, this technique uses gel electrophoresis to separate native or denatured proteins by the length of the polypeptide (denaturing conditions) or by the 3-D structure of the protein (native/ non-denaturing conditions).
Other related techniques include using antibodies to detect proteins in tissues and cells by immune staining and enzyme-linked immune sorbent assay (ELISA). This method originated from the laboratory of George Stark at Stanford. The name Western blot was given to the technique by W. Neal Burnette and is a play on the name Southern Blot, a technique for DNA detection developed earlier by Edwin Southern.
It is an analytical method wherein a protein sample is electrophoresed on an SDS-PAGE and electro transferred onto nitrocellulose membrane. The transferred protein is detected using specific primary antibody and secondary enzyme labelled antibody and substrate.
In this technique first of all the sample of proteins is separated on the basis of their molecular mass using SDS-PAGE or two-dimensional electrophoresis. Electrophoresis moves the proteins from the gel and onto the nitrocellulose where proteins adhere.
To detect a specific protein, an antibody to that protein must be available. The nitrocellulose membrane itself has many non-specific sites that can bind proteins, including antibodies which must be blocked with a non-specific protein solution, such as re-hydrated powdered milk.
The primary antibody is added in the milk solution and binds to the protein of interest. The antibody protein complex is detected using a secondary antibody that has a label attached to it (Fig. 3.27). Often a reporter enzyme such as alkaline phosphatase is linked to the secondary antibody, and the addition of lumiphos or X-phos to the blot allows detection of the protein band.
Information Obtained from Western Blotting:
Western blot gives you information on the following:
a. Size of protein
b. Expression amount of protein.
Uses of Western Blotting:
Western blotting is mostly used as a medical diagnostic technique. A positive Western blot can usually confirm an HIV infection. The confirmatory HIV test employs a Western blot to detect anti-HIV antibody in a human serum sample.
A Western blot is also used as the definitive test for Bovine spongiform encephalopathy (BSE, commonly referred to as ‘mad cow disease’). Western blotting is also helpful in the diagnosis of some forms of Lyme disease.
Technique # 4. South-Western Blotting:
South-western blotting is the combination of Southern blotting and Western blotting. It was first described by Bowen et al. and was used to identify DNA-binding proteins that specifically interact with a chosen DNA fragment in a sequence-specific manner.
In this technique, mixtures of proteins such as crude nuclear extracts or partially purified preparations are first fractionated on a sodium dodecyl sulfate (SDS) denaturing gel; the gel is then equilibrated in a SDS-free buffer to remove detergent and the proteins transferred by electro blotting to an immobilizing membrane.
During the transfer, proteins re-nature and hence DNA-binding proteins may subsequently be detected on the membrane by their ability to bind radio-labelled DNA.
Fractionation of crude nuclear extracts on an SDS gel followed by electro blotting and analysis for sequence-specific DNA binding directly on the blot combines the advantages of a high-resolution fractionation step with the ability to rapidly analyse for a large number of different DNA-binding specificities.
Technique # 5. North-Western Blotting:
North-western blotting technique is the combination of northern blotting and western blotting. This technique is used for identification of protein-RNA interactions in which protein is run on a gel, blotted, and probed with a labelled RNA of interest. Interactions are detected as hot-spots on the filter.
Technique # 6. Dot Blotting:
It is a modified version of Western blotting which is used for identification and analysis of protein of interest. Dot blot methodology differs from traditional Western blot techniques by not separating protein samples using electrophoresis. Sample proteins are instead spotted onto membranes and hybridized with an antibody probe.
Technique # 7. Zoo Blotting:
Zoo blotting is based on the principles of Southern blotting. In the genome of any organisms there are two regions – the coding and non- coding regions. It is the coding region that causes interest for most of the researchers as it is associated with the genetic information for a specific protein.
But the problem is that most of the regions of DNA are non-coding. The question is how to identify coding regions in large amount of non-coding DNA.
Zoo blotting is precisely .Used to distinguish coding DNA from non-coding regions. During evolution, the base sequence of non-coding DNA mutates and changes rapidly, whereas coding sequences change much more slowly and can still be recognized after millions of years of divergence between two species.
Therefore, DNA is extracted from a series of related animals, such as a human, monkey, mouse, hamster, cow, etc. Samples in this DNA “zoo” are each cut off with a suitable restriction enzyme and the fragments are run on a gel and transferred to a nylon membrane. They are probed using DNA that is suspected of being human coding DNA.
If a DNA sample really does include a coding sequence, it will probably hybridize with some fragment of DNA from most other closely related animals (Fig. 3.29). If the DNA is non-coding DNA, it will probably hybridize only to the human DNA.