Nucleic acid hybridization is a basic technique in molecular biology which takes advantage of the ability of individual single-stranded nucleic acid molecules to form double-stranded molecules. According to Watson-Crick base pairing, adenine binds with thymine and guanine binds with cytosine by hydrogen bonding.
The basic principle behind the southern hybridization is the nucleic acid hybridization. Southern hybridization commonly known as southern blot is a technique employed for detection of a specific DNA sequence in DNA samples that are complementary to a given RNA or DNA sequence. It was the first blotting technique to be devised, named after its pioneer E.M Southern, a British biologist. Southern blotting involves separation of restricted DNA fragments by electrophoresis and then transferred to a nitrocellulose or a nylon membrane, followed by detection of the fragment using probe hybridization.
Separated by electrophoresis is transferred from gel to a membrane which in turn is used as a substrate for hybridization analysis employing labeled DNA or RNA probes specific to target fragments in the blotted DNA. Southern hybridization helps to detect specific fragment against a background of many other restriction fragments. Southern blotting is a technique which is used to confirm the identity of a cloned fragment or for recognition of a sub-fragment of interest from within the cloned DNA, or a genomic DNA. Southern blotting is a prerequisite to techniques such as restriction fragment length polymorphism (RFLP) analysis.
- Southern blotting is an example of RFLP (restriction fragment length polymorphism). It was developed by Edward M. Southern (1975). Southern blotting is a hybridization technique for identification of particular size of DNA from the mixture of other similar molecules. This technique is based on the principle of separation of DNA fragments by gel electrophoresis and identified by labelled probe hybridization.
- Basically, the DNA fragments are separated on the basis of size and charge during electrophoresis. Separated DNA fragments after transferring on nylon membrane, the desired DNA is detected using specific DNA probe that is complementary to the desired DNA.
- A hybridization probe is a short (100-500bp), single stranded DNA. The probes are labeled with a marker so that they can be detected after hybridization.
- Restriction digest: by RE enzyme and amplification by PCR
- Gel electrophoresis: SDS gel electrophoresis
- Denaturation: Treating with HCl and NaOH
- Baking and Blocking with casein in BSA
- Hybridization using labelled probes
- Visualization by autoradiogram
Step I: Restriction digest
- The DNA is fragmentized by using suitable restriction enzyme. RE cuts the DNA at specific site generating fragments
- The number of fragments of DNA obtained by restriction digest is amplified by PCR
Step II: Gel electrophoresis
- The desired DNA fragments is separated by gel electrophoresis
Step III: Denaturation
- The SDS gel after electrophoresis is then soaked in alkali (NaOH) or acid (HCl) to denature the double stranded DNA fragments.
- DNA strands get separated
Step IV: Blotting
- The separated strands of DNA is then transferred to positively charged membrane nylon membrane (Nitrocellulose paper) by the process of blotting.
Step V: Baking and blocking
- After the DNA of interest bound on the membrane, it is baked on autoclave to fix in the membrane.
- The membrane is then treated with casein or Bovine serum albumin (BSA) which saturates all the binding site of membrane
Step VI: Hybridization with labelled probes
- The DNA bound to membrane is then treated with labelled probe
- The labelled probe contains the complementary sequences to the gene of interest
- The probe bind with complementary DNA on the membrane since all other non-specific binding site on the membrane has been blocked by BSA or casein.
Step VII: Visualization by Autoradiogram
- The membrane bound DNA labelled with probe can be visualized under autoradiogram which give pattern of bands.
Southern blotting is the transfer of DNA fragments from an electrophoresis gel to a membrane support, resulting in immobilization of the DNA fragments, so the membrane carries a semipermanent reproduction of the banding pattern of the gel. After immobilization, the DNA can be subjected to hybridization analysis, enabling bands with sequence similarity to a labeled probe to be identified. This unit describes Southern blotting via upward capillary transfer of DNA from an agarose gel onto a nylon or nitrocellulose membrane, and subsequent immobilization by UV irradiation (for nylon) or baking (for nitrocellulose). A Support Protocol describes how to calibrate a UV transilluminator for optimal UV irradiation of a nylon membrane. An alternate protocol details transfer using nylon membranes and an alkaline buffer, and is primarily used with positively charged nylon membranes. A second alternate protocol describes a transfer method based on a different transfer-stack setup. The traditional method of upward capillary transfer of DNA from gel to membrane has certain disadvantages, notably the fact that the gel can become crushed by the weighted filter papers and paper towels that are laid on top of it. This slows down the blotting process and may reduce the amount of DNA that can be transferred. The downward capillary method described in the second alternate protocol is therefore more rapid and can result in more complete transfer.
Application of Southern blotting:
- Southern blotting technique is used to detect DNA in given sample.
- DNA finger printing is an example of southern blotting.
- Used for paternity testing, criminal identification, victim identification
- To isolate and identify desire gene of interest.
- Used in restriction fragment length polymorphism
- To identify mutation or gene rearrangement in the sequence of DNA
- Used in diagnosis of disease caused by genetic defects
- Used to identify infectious agents
Northern blotting was developed by James Alwine, George Stark and David Kemp (1977). Northern blotting drives its name because of its similarity to the first blotting technique, which is Southern blotting, named after the biologist Edwin Southern. The major difference is that RNA being analyzed rather than DNA in the northern blot.
Expression of a particular gene can be detected by estimating the corresponding mRNA by Northern blotting. Northern blotting is a technique where RNA fragments are separated by electrophoresis and immobilized on a paper sheet.Identification of a specific RNA is then done by hybridization using a labeled nucleic acid probe. It helps to study gene expression by detection of RNA (or isolated mRNA) in a sample.
In Northern blotting, probes formed of nucleic acids with a sequence which is complementary to the sequence or to a part of the RNA of interest. The probe can be DNA, RNA or chemically synthesized oligonucleotides of minimum 25 complementary bases to the target sequence.
Fig 3-4.3: Steps of Northern Hybridization
The northern blotting involves the following steps:
1. Total RNA is extracted from a homogenized tissue sample or cells. Further eukaryotic mRNA can then be isolated by using of oligo (dT) cellulose chromatography to isolate only those RNAs by making use of a poly A tail.
2. The isolated RNA is then separated by gel electrophoresis.
3. The RNA samples separated on the basis of size are transferred to a nylon membrane employing a capillary or vacuum based system for blotting.
Fig 3-4.3.1: Setup for Northern blotting
4. Similar to Southern blotting, the membrane filter is revealed to a labeled DNA probe that is complementary to the gene of interest and binds.
5. The labeled filter is then subjected to autoradiography for detection.
The net amount of a specific RNA in a sample can be estimated by using Northern blot. This technique is widely used for comparing the amounts of a particular mRNA in cells under different conditions. The separation of RNA samples is often done on agarose gels containing formaldehyde as a denaturing agent as it limits the RNA to form secondary structure.
Analysis of Northern Blot:
RNA extract is electrophoresed in an agarose gel, using a denaturing electrophoresis buffer (containing formaldehyde) to ensure that the RNAs do not form inter- or intra-molecular base pairs, as base pairing would affect the rate at which the molecules migrate through the gel. After electrophoresis, the gel is blotted onto a nylon or nitrocellulose membrane, and hybridized with a labeled probe. If the probe is a cloned gene, the band that appears in the autoradiograph is the transcript of that gene. The size of the transcript can be determined from its position within the gel, and if RNA from different tissues is run in different lanes of the gel, then the possibility of differentially expressed gene can be examined.
Northern blotting helps in studying gene expression pattern of various tissues, organs, developmental stages, pathogen infection, and also over the course of treatment. It has been employed to study overexpression of oncogenes and down-regulation of tumor-suppressor genes in cancerous cells on comparison with healthy tissue, and also for gene expression of immune-rejection of transplanted organ.
The examination of the patterns of gene expressions obtained under given conditions can help determine the function of that gene.
Northern blotting is also used for the analysis of alternate spliced products of same gene or repetitive sequence motif by investigating the various sized RNA of the gene. This is done when only probe type with variation in one location is used to bind to the target RNA molecule.
Variations in size of a gene product may also help to identify deletions or errors in transcript processing, by altering the probe target that can be used along the known sequence and make it possible to determine the missing region of the RNA.
It is a rapid method of isolating a colony containing a plasmid harboring a particular sequence or a gene from a mixed population. The colonies to be screened are first replica-plated on to a nitrocellulose filter disc that has been placed on the surface of an agar plate prior to inoculation. Master plate is retained for reference set of colonies. The filter bearing the colonies is removed and treated with alkali so that the bacterial colonies are lysed and the DNA they contain is denatured. The filter is then treated with proteinase K to remove protein and leave denatured DNA bound to the nitrocellulose. The DNA is fixed firmly by baking the filter at 80°C. A labeled probe is hybridized to this DNA which is monitored by autoradiography. A colony whose DNA print gives a positive auto radiographic result on X-ray film can then be picked from the reference plate.
Colony hybridization can be used to screen plasmid or cosmid based libraries.
Fig 3-4.4: Colony hybridization
n Situ Hybridization (ISH)
It is a technique that employs a labeled complementary nucleotide strand (i.e. probe) for localizing specific DNA or RNA sequence targets within fixed tissues and cells (i.e in situ). Probes used for hybridization can be double-stranded DNA probes, single-stranded DNA probes, RNA probes, synthetic oligonucleotides. There are two ways available to detect DNA or RNA targets
Chromogenic (CISH) in situ hybridization and Fluorescence in situ hybridization (FISH).
Chromogenic in situ hybridization (CISH)
It uses the labeling reactions involving alkaline phosphatase or peroxidase reactions to visualize the sample using bright-field microscopy. It is primarily used in molecular pathology diagnostics. CISH can also be employed for samples like fixed cells or tissues, blood or bone marrow smears and metaphase chromosome spreads.
- Western blotting technique is used for identification of particular protein from the mixture of protein.
- In this method labelled antibody against particular protein is used identify the desired protein, so it is a specific test. Western blotting is also known as immunoblotting because it uses antibodies to detect the protein.
- Extraction of protein
- Gel electrophoresis: SDS PAGE
- Blotting: electrical or capillary blotting
- Blocking: BSA
- Treatment with primary antibody
- Treatment with secondary antibody( enzyme labelled anti Ab)
- Treatment with specific substrate; if enzyme is alkaline phosphatase, substrate is p-nitro phenyl phosphate which give color.
Step I: Extraction of Protein
- Cell lysate is most common sample for western blotting.
- Protein is extracted from cell by mechanical or chemical lysis of cell. This step is also known as tissue preparation.
- To prevent denaturing of protein protease inhibitor is used.
- The concentration of protein is determined by spectroscopy.
- When sufficient amount of protein sample is obtained, it is diluted in loading buffer containing glycerol which helps to sink the sample in well.
- Tracking dye (bromothymol blue) is also added in sample to monitor the movement of proteins.
Step II: Gel electrophoresis
- The sample is loaded in well of SDS-PAGE Sodium dodecyl sulfate- poly-acrylamide gel electrophoresis.
- The proteins are separated on the basis of electric charge, isoelectric point, molecular weight, or combination of these all.
- The small size protein moves faster than large size protein.
- Protein are negatively charged, so they move toward positive (anode) pole as electric current is applied.
Step III: Blotting
- The nitrocellulose membrane is placed on the gel. The separated protein from gel get transferred to nitrocellulose paper by capillary action. This type of blotting is time consuming and may take 1-2 days
- For fast and more efficient transfer of desired protein from the gel to nitrocellulose paper electro-blotting can be used.
- In electro-blotting nitrocellulose membrane is sandwich between gel and cassette of filter paper and then electric current is passed through the gel causing transfer of protein to the membrane.
Step IV: Blocking
- Blocking is very important step in western blotting.
- Antibodies are also protein so they are likely to bind the nitrocellulose paper. So before adding the primary antibody the membrane is non-specifically saturated or masked by using casein or Bovine serum albumin (BSA).
Step V: Treatment with Primary Antibody
- The primary antibody (1° Ab) is specific to desired protein so it form Ag-Ab complex
Step VI: Treatment with secondary antibody
- The secondary antibody is enzyme labelled. For eg. alkaline phosphatase or Horseradish peroxidase (HRP) is labelled with secondary antibody.
- Secondary antibody (2° Ab) is antibody against primary antibody (anti-antibody) so it can bind with Ag-Ab complex.
Step VII: Treatment with suitable substrate
- To visualize the enzyme action, the reaction mixture is incubated with specific substrate.
- The enzyme convert the substrate to give visible colored product, so band of color can be visualized in the membrane.
- Western blotting is also a quantitative test to determine the amount of protein in sample.
- To determine the size and amount of protein in given sample.
- Disease diagnosis: detects antibody against virus or bacteria in serum.
- Western blotting technique is the confirmatory test for HIV. It detects anti HIV antibody in patient’s serum.
- Useful to detect defective proteins. For eg Prions disease.
- Definitive test for Creutzfeldt-Jacob disease, Lyme disease, Hepatitis B and Herpes.