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

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