Paternity issues increased since a few decades back. Before the advent of recombinant DNA technology, it was difficult to recognize the alleged parents of an individual. However, with the help of DNA technology, it is now easy to find out the relationship of an individual. Not only the parents but also the individual’s close relatives get identified. In recent years, DNA testing became accessible to people with accurate results. DNA technology enables the discovery of paternity or maternity of an individual. It is useful for adoption, child support, and immigration issues. Sir Alec Jeffreys developed the process of DNA profiling or DNA typing for the first time. It is a commonly used technique in crime scene investigation. DNA typing has revolutionized forensic science since it traces the DNA of the suspect or the criminal.

Who is the father of the child?

Imagine a fictional scenario. A woman blames a man for being her child’s father. The alleged individual does not accept it. Such a case gets dragged to the court of justice and gets forwarded to DNA analysis.

No two individuals possess the same genome. Every individual’s genome slightly differs. However, using DNA polymorphisms help in analyzing the DNA. DNA typing or DNA fingerprinting technique helps to detect the paternity issues. DNA typing is an individual-specific autoradiography technique largely involving banding procedures. DNA digestion involves treatment with a restriction endonuclease that cleaves outside a family of VNTRs. It also involves a southern blot. Paternity testing involves obtaining the samples from the mother, the alleged father, and the child. Sources of DNA include buccal swab, blood, saliva, semen, toothbrush, razor, sperms, vaginal lubrication or another appropriate fluid source.

DNA profiling works on the principle of inheritance. The fusion of the male and female gametes results in the formation of the zygote. The zygote gets one-half DNA from the mother and the other half from the father. If the father’s DNA markers match half of the child’s markers, the real identity of the child’s father gets revealed.

Image: DNA typing procedure

The procedure starts with the collection of the samples from the mother, the alleged father, and the child. The collection of samples in three different tubes is followed by labeling the tubes with appropriate information. Isolated DNA from the blood cells gets processed further.

This process involves cutting the DNA using restriction enzymes. These enzymes cleave at a specific site known as the restriction site. The process of cleaving the DNA into fragments is known as restriction digestion. The cut fragments get analyzed using a marker.

The DNA fragments get separated using electrophoresis. The procedure of electrophoresis involves the addition of the mother’s, alleged father’s, and the child’s DNA samples into the electrophoretic wells. The test samples get compared with the standard samples. Due to the electric field, the negatively charged molecules such as DNA move towards the positive poles. The gel used in the electrophoretic technique mostly involves an agarose gel. The smaller fragments of the DNA travel faster. Hence, it is possible to separate the fragments based on their sizes. Staining the gel with the ethidium bromide helps in visualizing the bands. The DNA fragments are visible in the form of bands under UV light.

The electrophoretic gel gets transferred to the membrane filter by Southern blotting technique. The southern blotting apparatus involves many things. The gel gets placed in a tray filled with the alkaline buffer solution. A blotting paper helps the buffer to move towards the membrane filter. The membrane filter placed on the gel is known as the nitrocellulose filter. The placement of the paper towels and a weight on top of the membrane filter fix the membrane in one place. The DNA present on the gel interacts with the buffer and travels to the membrane filter. The DNA binds to the nitrocellulose filter. Then the DNA present on the membrane gets treatment with the probes. The nitrocellulose membrane filter exactly shows the DNA fragments as separated on the gel.

Then the DNA gets fixed permanently on the membrane by heating at 80 degrees C for 2-3 hours. Now the DNA on the membrane gets completely hybridized with the probes. The probe forms a complementary base pair with the homologous DNA sequence. DNA fragments get probed with STR or VNTR probes. The probe binds to the specific DNA sequences on the filter. Next step involves washing the unbound probes with an appropriate solution. Hence, only the hybridized radioactive probes get retained on the filter.

It is a process in which the probed DNA gets exposure of a photographic emulsion forming a pattern on the film. The banding pattern thus obtained for each sample is known as a DNA fingerprint.

Detection of two DNA fragments for every individual is the key to interpret the data. The DNA fragments have a specific size and denote heterozygosity for a particular pair of alleles. If one of the fragments of the baby’s DNA matches with the mother and the alleged father, it indicates the paternity of the alleged person. An early technique of DNA typing is known as RFLP technique. However, this technique needs the support of other techniques such as PCR technique.

Polymerase chain reaction mimics the process of DNA replication and helps in amplifying the DNA. Identification of the biological parents of the child uses this method. It is a relatively convenient method and takes less time.

Minute percentage of DNA involves a different sequence in every individual. It decides the factors such as variation, mutation, and others. It accounts for just 0.1 percentages.

Table: Information on genome and DNA typing

It is convenient and easy to obtain buccal samples. No need

for

invasive techniques. The technique maintains privacy since the test result never gets shared with anyone except the patient or the individual who needs the information. Y-STR involves the resolution of a mixed DNA sample from the male and female respectively. If the child is a male, Y-STR helps a lot. The Y chromosome is known as a sex chromosome. Hence, it is useful in determining paternity. DNA typing studies also utilize mitochondrial DNA. The mtDNA follows the

maternal

pattern of inheritance. Thus, mtDNA analysis also works well in the DNA profiling.





References:

[1]

Fundamentals of Forensic DNA Typing,

John M. Butler

[2]

Forensic DNA Analysis,

Lawrence F. Kobilinsky, Louis Levine, Henrietta Margolis-Nunno

[3]

Recombinant DNA Technology,

Keya Chaudhuri



