Southern blotting technique

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

[2] Ana Techniques in Biotechnology, Goutam Bhowmik
[3] Gene Cloning and DNA Analysis, T.A. Brown

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Molecular testing helps to detect gene mutations

The basic unit of the heredity known as the gene plays a crucial role in disease and inheritance. If it gets mutated at wrong sites, it passes on the undesired traits to the population. Genetic diseases arise due to an underlying mutation in the gene. The effect of all mutations may not necessarily be a disease. However, the mutations occurring at wrong sites, lead to altered products such as proteins and enzymes having altered functions leading to genetic disorders. There are many examples of genetic disorders in humans. They follow different patterns of inheritance. For example, the BRCA gene mutation leads to breast cancer. Similarly, mutations in the mitochondrial DNA follow the maternal pattern of inheritance.

Image 1: Molecular testing

The genetic disorders are either present by birth or may develop during later stages of life. Most of them are congenital, meaning, their occurrence is by birth. With the help of genetic testing, it is possible to detect them before birth. Importance of genetic testing lies in the prevention of genetic disorders. It helps in preventing the birth of babies who may suffer later on. It helps in decision making and genetic counseling. Genetic testing also reveals types of mutations. We can understand the chances of getting a disease in the future. Thus, the field of genetic testing is advancing further. With the help of genetic testing, it is possible to detect disease genes in a family.

The field of diagnostics involves detecting a disease, the stages of the disease, and the causative agent. Genetic testing helps to detect the gene involved in mutation, the type of mutation, chances of inheriting a disease, and whether the disease will occur in the future. It mainly targets a population with a family history of diseases. The purpose of genetic testing is to detect any mutation before the birth of the baby. There are three important applications of genetic testing such as prenatal diagnosis, newborn screening, and carrier detection. Prenatal diagnosis checks whether the fetus is at risk. Two important procedures involved in prenatal diagnosis are amniocentesis and chorionic villus sampling. These two procedures being very painful and risky led to the development of non-invasive prenatal testing (NIPT). It involves testing cell-free DNA that floats in the maternal plasma. The prenatal tests can detect gene mutations and chromosomal abnormalities. The embryos containing mutated genes lead to serious genetic diseases. Genetic testing helps to detect mutations in the embryonic genes. Newborn screening detects mutations in the newborn babies.

For example, the PKU test detects mutations for phenylalanine that causes phenylketonuria. Carrier detection helps to find out whether the individuals are carriers for a gene mutation. The example involves sickle cell anemia. The carriers are known as heterozygotes. DNA molecular testing determines the molecular nature of mutations associated with the disease.

Molecular testing methods include RFLP and PCR Analysis:

1.     RFLP Analysis:

It is a commonly used genetic test. It involves analyzing restriction fragment length polymorphisms. It arises due to a different pattern of restriction sites. The RFLP analysis involves three main steps such as restriction digestion, agarose gel electrophoresis, and hybridization. The DNA is fragmented using restriction enzymes. The fragments obtained from restriction digestion are separated based on the fragment size using AGE. The gel containing separated fragments is placed in a buffered solution to transfer the DNA to a membrane filter. The DNA on the membrane filter hybridizes with the probe having a complementary sequence.

The product of restriction endonuclease action on a DNA is known as a restriction digest. The restriction fragment length polymorphism is nothing but a variant in DNA banding pattern of an electrophoresed restriction digest. It occurs when the length of the fragment varies between the individuals. Each length of a fragment is an allele. The DNA shows restriction enzyme cleavage site at a place in the genome of an individual which is missing is another individual.

Image 2: RFLP analysis

An example of RFLP analysis involves detection of sickle cell gene by Dde I RFLP. Abnormal hemoglobin results into sickling of red blood cells in sickle cell anemia. It arises due to a beta-globin gene mutation. It involves a single base-pair change of AT to TA leading to single nucleotide polymorphisms. The sixth codon of the beta-globin thereby changes from GAG to GUG. Instead of glutamic acid insertion in a polypeptide, valine gets added. This mutation results in RFLP for restriction enzyme Dde I. This enzyme has a restriction site where the fourth base pair changes.

In normal individuals, there are three Dde I sites in the beta-globin genes. One of the Dde I sites is situated upstream while the other two sites are present in the coding regions. In the individuals having sickle cell anemia, the beta-globin genes have only two Dde I sites instead of three. The mutation removes the Dde I site present in the middle. RFLP analysis helps to study individuals affected with sickle cell disease and compare the results with the normal individuals.

In a normal individual, the fragments hybridized with the probes reveal two fragments after visualization. One fragment shows 175 base pair size. The other fragment shows 201 base pair size. In persons with sickle cell disease, the hybridized DNA, when visualized, reveals only one fragment of 376 base pair.  The DNA obtained from heterozygotes reveals three bands such as bands with 376, 201 and 175 base pairs respectively. Let us consider another example involving mutations in the DNA flanking the gene. The DNA flanking the gene may be very far away from such as PKU genes. In such cases, the detection of mutation relies on flanking RFLP.

2.     PCR Analysis:
     

Image 3: Polymerase chain reaction

    For PCR analysis, the sequence information helps in designing the primers. Polymerase chain reaction or PCR is nothing but amplifying the desired DNA under controlled conditions. A common test involved in the PCR analysis is Allele-specific oligonucleotide hybridization (ASO). A short single-stranded DNA capable of being synthesized in a test tube is known as an oligonucleotide. It is less than 50 nucleotides in length. These small DNA molecules are capable of hybridizing. They form a complementary base pair with other DNA molecules. A single mismatch may not allow an oligonucleotide to hybridize, thereby discriminating between the two alleles of an SNP.

Consider an example of ASO hybridization. The GLC1A gene mutations lead to ophthalmic disorders. A common example includes open-angle glaucoma. It is a condition in which there is an increase in eye pressure. It may lead to total blindness due to neglection. One of the GLC1A gene mutations involves CG to TA change. Thus the codon changes from CCG (encoding proline) to CUG (encoding leucine). The DNA sequencing of the heterozygotes revealed both wild-type and mutant alleles. Agarose gel electrophoresis helps to separate the fragments. The DNA denatures into single strands for ASO hybridization. The detection becomes easy through radioactive signaling. There is one more concept associated with ASO. It is known as reverse ASO hybridization. The method uses radioactively labeled PCR products as probes for hybridization with many different ASOs. It can detect many mutations.

Sometimes it might be difficult to find some genes in the genome. The gene may be cloned but not sequenced. Molecular testing tools may not be available for such genes. Sometimes a single molecular test may be futile if the gene has many mutations. RFLP and PCR can better detect single base pair mutations or genes with only a few mutations. In case of multiple mutations, designing of a subset for known mutations may help.

References:
[1] RFLP analysis, Thermofisher Scientific
[2] Molecular Biology Techniques: An Intensive Laboratory Course, Walt Ream, Katharine G. Field
[3] Ana Techniques in Biotechnology, Goutam Bhowmik

© Copyright, 2018 All Rights Reserved.