Mitochondrial DNA and Human Ancestry

Mitochondria are important organelles for generating energy in the cells. They have a unique genome. In some species, the mitochondrial DNA is linear. Whereas in some species it is circular. The endosymbiont theory revealed that the mitochondria and chloroplasts originated as prokaryotes. Later on, they invaded the eukaryotic cells to establish a symbiotic relationship. Mitochondria are involved in cellular respiration which is a process of oxidization of food molecules. Mainly, the oxidization of glucose gives rise to carbon dioxide and water. The energy released in this process is known as ATP (Adenosine Triphosphate). Thus, mitochondria are also known as energy powerhouses. Till now we only knew the genes present in the nucleus. We have studied the structure and expression of the nuclear genes. Years later the discovery of the nuclear genes, the scientists found that there was something else beyond the Mendelian pattern of inheritance. Mitochondria also controlled a few traits. The genes present in the cytoplasmic organelles such as mitochondria and chloroplast are known as extra-nuclear genes. They are alternatively known as Non-Mendelian genes because they do not follow Mendelian inheritance pattern.

Image 1: Mitochondrion and its DNA

The Mitochondrial Genome:

The origin of the mitochondrial DNA is of bacterial or prokaryotic origin. Thus, it shows similar replication and gene expression features. A wide structural variation persists in the eukaryotic mitochondrial genome. The complete sequences of mitochondrial DNA are now known to us. The structure of the mitochondrial genome is highly conserved in the mammals. Though mitochondria have their separate genomes, they are not completely self-operative. In some cases, they may also require nuclear gene products for carrying out their specific functions. The copy number of the mitochondrial genome in the oocytes is extremely high. Fertilization is a process in which the sperm and the egg fuse with each other. During this process, the mitochondria present in the sperm do not get transferred to the oocyte. Thus, the zygote contains the mitochondria of the oocyte. Thus it follows a maternal pattern of inheritance.

Description of mitochondrial DNA:

The mitochondrial DNA is double-stranded, supercoiled and circular. The mitochondrial DNA is also known as mtDNA. It codes for 37 genes in humans, and 35 genes in S. cerevisiae. The mitochondrial DNA consist of 16.6 kilobases nucleotide base pairs. The GC content of mtDNA differs from that of the nuclear DNA. Many genes in the mitochondria encode for the enzymes involved in the oxidative phosphorylation. Extranuclear processes require many such enzymes. For some of the processes, the nuclear genes may not be sufficient to encode proteins or enzymes. Thus, mitochondrial genes play a crucial role in synthesizing enzymes that catalyze reactions for generation of energy.

An abnormality in the mitochondrial genes may lead to mitochondrial disorders. The mitochondrial DNA does not associate with the histone proteins. There is very little extragenic or repetitive DNA in the mitochondrial genome. The human mitochondrial genome is small and compact. The genes are very close to each other with a very little space between them. In yeast, the mitochondrial genome consists of widely spaced genes. Mitochondrial genomes are studied using metagenomics.

Just like the nuclear DNA, the mitochondrial DNA also undergoes a semi-conservative type of replication. Mitochondrial DNA also requires DNA polymerases to carry out the process of replication. These enzymes are known as mitochondrial DNA polymerases. The mtDNA replication occurs throughout the cell cycle. Unlike nuclear DNA replication, the mtDNA is not S-phase specific. The displacement loop or D-loop model is studied. The two mtDNA strands show different densities. The bases are spaced unequally on the strands. The unequal spacing or distribution of bases creates heavy and light strands. Letter ‘H’ is used to denote the heavy strand. Letter ‘L’ is used to denote the light strand of the mitochondrial DNA. The mtDNA is supercoiled up to a hundred coils. The uncoiling takes place during the initiation of replication. The synthesis of H strand starts at the origin of replication. Formation of a ‘D’ shaped loop takes place. The loop expands further to synthesize a new light strand. Synthesis of the light strand starts at the second replication origin. Thus, mitochondrial DNA follows a continuous type of replication for both the strands. After completion of the replication, the supercoiling of the two circular mtDNAs occurs. The heavy and light strands consist of mitochondrial protein-coding genes.

Image 2: Mitochondrial inheritance

Investigation of the genetic relationships:

Mitochondria exclusively follow maternal inheritance. It means that they are inherited only from the female gamete, the oocyte. Thus, the oocyte is the major contributor of the cytoplasm. The mitochondrial DNA analysis can be used to assess the genetic variability of a given population. The diseases caused due to maternal inheritance get detected using information related to mitochondrial DNA mutations. Finding the ancestors and relatives of an individual may be easier.

The story of the Tsar and Tsarina:

The Bolsheviks executed the Tsar and his family in the eighteenth century. However, one of the Tsar’s three daughters, Princess Anastasia managed to escape from the execution. After many years, a woman claimed herself as Anastasia. She lived in the United States for many years as Anna Anderson. The Tsar’s family members’ mtDNA samples were used to study their relationships. Thus mtDNA analysis helped to reveal the true relationships in the Romanovs.

Human ancestry linked to mtDNA studies:

Various studies revealed that genetic recombination is uncommon in the mitochondrial DNA. Thus it is similar to the Y chromosome. The mtDNA is a good genetic marker for tracing the human ancestry. Human mitochondrial DNA undergoes evolutionary changes in the sequences at an approximately constant rate. The statistical figures suggest one change per mitochondrial lineage every three thousand to four thousand years. Analyzing mitochondrial DNA from human bones provides a good number of lineal history evidence. Just as the Y chromosome is inherited only through the father, the mitochondrial DNA is inherited only through the mother. Thus mitochondrial DNA exhibits itself as a family album with pictures of the members. It helps to identify the degree of kinship and the familial origin.

Various studies report a pre-historic migration of human population into North America. The first mtDNA study revealed that Native Americans descended from the Asian ancestors many years ago. They found four distinct mitochondrial haplogroups. Another study revealed the African origin of the modern human population. You might know the story of an African woman (called as Mitochondrial Eve), who lived many lakhs of years ago. She passed on her mitochondrial DNA to future generations. Similarly, the man who passes on the Y chromosome was known as Y chromosome- Adam. Hence, human origin is a topic of debate.

The sequences from the bones of the Neandertal-human were analyzed. PCR analysis was used to obtain DNA sequences from the bone specimens. The relationships between the Neandertals and modern humans were studied using phylogenetic trees. Molecular information such as mtDNA sequences and immunological data were studied. Thus, mitochondrial DNA provided a crucial role in tracing the human ancestry and origin.

References:

[1] Medical genetics, G.P. Pal

[2] Mitochondrial DNA- Wikipedia

[3] Genomics of Chloroplasts and Mitochondria, Ralph Bock, Volker Knoop

[4] Introduction to Genomics, Arthur Lesk
[5] Biomolecular Archaeology: An Introduction, T. A. Brown, Keri Brown

© Copyright, 2018  All Rights Reserved

To Study the Role of Telomerase in Cellular Replication

An introduction to telomeres:

A telomere is an important structure of the chromosome. Every chromosome consists of a special DNA-protein complex with a particular sequence. Thus telomeres are the caps at the end of the chromosomes. A human telomere is 3 to 20 kilobases in length. It consists of tandem repeats of TTAGGG-3’ sequence. The telomeric sequence is 3000 times repeated. When you go out on a sunny day, would you not prefer to wear a cap? Sun’s scorching heat would be unbearable and wearing a cap would protect you from the damaging heat. Similarly, telomeres are caps on the chromosomes.

 There are three important functions of telomeres:

1.     Telomeres provide structural stability to the chromosomes by sealing their ends.

2.     They protect the ends of the chromosomes from damage or formation of rings.

3.     Telomeres protect the chromosomes from fusing with other DNA.

A telomerase enzyme is a ribonucleoprotein complex. It acts like a reverse transcriptase. It plays a crucial role in terminal chromosomal maintenance. Telomerase adds telomeric repeats to the chromosomal ends or DNA termini. A telomerase enzyme compensates for incomplete replication. Telomerase can elongate the telomeres. They enable the cells to distinguish between natural chromosomal ends and double-stranded breaks. They maintain chromosome stability.

Species	Telomere repeat sequence	Telomerase RNA template sequence
Human	5’-TTAGGG-3’	5’-CUAACCCUAAC-3’
Oxytricha	5’-TTTTGGGG-3’	5’-CAAAACCCCAAAACC-3’
Tetrahymena	5’-TTGGGG-3’	5’-CAACCCCAA-3’

Table: The telomeric repeat sequences and telomerase RNA template sequences in humans, oxytricha, and tetrahymena respectively.

Structure of a telomere:

A telomere consists of the following components:

1.     DNA sequence:

A telomere consists of 5’TTAGGG 3’ sequence. A hexamer unit is present in 2000 copies.

2.     Loops

Several studies indicate the occurrence of telomeres in the form of loops. Most of the telomeres end in a loop known as T-loop, where the double-stranded telomeric tract curves around. The T-loop formation protects from exonucleases. There is a displacement loop of TTAGGG repeats known as a D loop. Telomeres can also form specialized structures known as G-quadruplex DNA. These structures are composed of guanine tetrads or G-quartets. Guanine tetrads are square planar arrays of four, hydrogen-bonded guanines. Hoogsteen base pairing is common among them. This G-quartets stack upon each other and provide telomere protection.

3.     Protein components

·        TRF1: It is known as a telomere repeat binding factor 1. It binds to the telomere at T-loop. TRF-1 inhibits telomerase-dependent elongation.

·        TRF 2: It is known as telomeric repeat binding factor 2. It is involved in the formation of T-loop. The overexpression of TRF2 in somatic cells leads to telomere shortening.

·    hRAP 1: It is a human homolog of yeast protein. It is involved in determining the length of the telomere.

·    TIN 2: It is known as a TRF-1 interacting nuclear factor. It promotes pairing of telomere repeats.

·        TANK1: It promotes telomere elongation.

Image 1: Chromosomes showing their telomeres

What is telomere shortening?

DNA replication is an important process of the cell. In this process, DNA is duplicated using replication enzymes. However, during DNA replication, the enzymes may skip replicating the ends of the DNA. As a result, few telomeric sequences get skipped. Thus, the replicated DNA is slightly shorter than the original. After many replications, the telomere sequences become too short. As a result, the cell division stops and the cell undergo apoptosis. This phenomenon leads to aging in human. The process of telomere shortening is observed in somatic cells and not in the germ cells, antibody-producing cells, and cells which constantly replace the gut epithelium. The telomerase enzyme is prominently present in these cells. That is why these cells do not undergo telomere shortening. Somatic cells are deficient in the telomerase enzyme.

There are two ways in which the telomere shortening occurs:

The extreme 3’ end of the DNA is difficult to copy. The natural position of the priming site may be beyond the end of the template. Thus, the lagging strand copy is incomplete, because the last Okazaki fragment is not complete. The resulting daughter molecule has a 3’ overhang and gives rise to a grand-daughter molecule which is shorter than the original one.

Another reason for a shortened telomere is the position of the primer. It is at an extreme 3’ end of the lagging strand.

The length of the telomere is a useful parameter in the process of aging.

What is telomere extension?

The telomerase contains a unique RNA-protein complex. The 5’ end of the telomerase consists of 5’-CUAACCCUAAC-3’ sequence. The central region of this sequence is a reverse complement of the telomere repeat. This repeat has a 5’- TTAGGG-3’ sequence.

The extension of telomeric DNA follows five steps. First, the telomerase RNA pairs with the ends of the molecule. Next, the telomeric DNA gets extended at a short distance. A stem-loop structure determines the length of the telomeric DNA. After extension of the telomeric DNA, the telomerase moves further through the translocation process. It starts base pairing the next fragment. In this way, the telomere gets extended.

The completion of telomere extension is unique. A new Okazaki fragment is primed and synthesized, which converts 3’ extensions into the complementary double-stranded ends. The t-loop is formed when a free 3’ end of telomere loops back and invades the double helix.

A process of replicative senescence takes place in the cells. The changes in the structure of the telomeres cause replicative senescence. Cellular senescence leads to changes in cell morphology and gene expression. It is triggered when cells acquire few critically short telomeres.

Eventually, the telomeres become very short. The telomere-protein complex gets disrupted and leads to DNA damage. P53 involved apoptosis is a similar kind of damage. Thus, for a normal senescent, further cell division is blocked.

The finite ability of telomeric DNA replication is known as the Hayflick limit.

Image 2: Telomere extension

Telomeres and cancer:

Most of the cancer cells have active telomerase enzyme. As a result, these cells keep on multiplying. The remaining cells employ an important mechanism known as alternative lengthening of telomeres (ALT) for telomere maintenance.

Most of the cancer cells contain telomerase, a crucial factor for the immortality of the cells.

Suppose there is a mutation in a gene controlling normal cell cycle arrest. Such a cell will divide in spite of having a very short telomere. This cell has the capability of becoming immortal. It may also carry plenty of mutations triggering to cancer.

“In sum, the primary cause of the cancer is a cellular mutation. Telomerase activity and telomere extension are secondary to cancer.”

What are anti-telomerase drugs?

Anti-telomerase drugs work against the telomerase. These drugs target the action of telomerase and block this enzyme. As a result, the cells do not undergo division. However, some side effects may occur due to the complete blocking of telomerase. Cells such as antibody-producing cells, the immune cells, germ cells, and other cells are dependent on telomerase. Thus there could arise some serious effects of using anti-telomerase drugs.

Conclusion:

More research has to be carried out before launching anti-telomerase drugs. Also, we must note that telomerase activity is not the only cause of cancer. Smoking, consumption of alcohol, pollution, chemical mutagens, and other factors may also contribute to the risk of cancer. Mainly these factors induce mutations in the cells. Thus, for designing a cancer drug, the focus should be on the underlying mutations.

References:

[1] Telomeres and Telomerase in Aging, Disease, and Cancer: Molecular Mechanisms, K. Lenhard Rudolph

[2] Genome Instability in Cancer Development, Erich A. Nigg

[3] Topics in Anti-Cancer Research, Atta-ur Rahman, Khurshid Zaman

[4] Concepts Of Genetics, 7/E (With Cd), Klug
[5] Molecular Biology of the Gene, 5th Ed, Pearson Education, 2004: Gene, Pearson Education, Inc

© Copyright, 2018  All Rights Reserved

Medical Genetics

Genetics plays an important role in the field of medical sciences. There arises a genetic base to a disease, the development, reproduction, and other processes in the body. The gene or a blueprint of life occurs in every cell and is an important inheriting factor. Hence, it is important to understand genes and their role in various diseases, cancers, developmental stages, and interactions with various components. Some of the genes encode for proteins. The genes get passed on from one generation to the next generation. They also carry any minute changes or mutations. The diverse population is a sign of variation. Due to recent advances in health research, more and more diseases showed a genetic base. The science of genetics helps in detecting many diseases. It helps in their treatment or prevention. When you try to understand the molecular intricacies of the genetic material, you will be amazed to see an entirely different world. Thousands of reactions keep occurring at a molecular level.

DNA, the blueprint of life consists of nucleotide base pairs attached to the phosphodiester bonds. Small units of DNA form genes and exhibit specific functions. Various disorders arise due to gene mutations. Hence, they are known as genetic disorders. Detection of genetic disorders involves modern genetic diagnostic techniques. There involves the tremendous importance of genetics in medical sciences. To understand its role in health and disease, we must know the basic concept of genetics. 

The review covers the following topics in detail:

1.     What are genes and various branches of genetics?

2.     Chromosomes and disorders associated with the genes

3.     Disorders inherited due to faulty genes

4.     The role of gene mutations in biochemical disorders

5.     Detection of genetic disorders

6.     Genetic counseling

7.     Gene Cloning and DNA analysis

8.     Gene therapy

Image 1: Medical genetics

Various branches of genetics:

Human genetics involves several branches such as cytogenetics, molecular genetics, biochemical genetics, cancer genetics, Immunogenetics, and developmental genetics. Cytogenetics is the study of chromosomes and various disorders associated with the same. A chromosome is a specialized structure consisting of DNA-protein complex packed in a condensed manner. Cytogenetic techniques help in studying these structures. Molecular genetics involves the study of genes at the molecular levels such as single base pair changes, mutations, and other studies. Biochemical genetics involves genes controlling the enzyme production. Cancer genetics involves studying cancer genes and mutations associated with cell cycle control. Different antigen-antibody interactions studies involve immunogenetics. Developmental genetics helps to study the genetic control of the development. Hence there are many such branches of genetics. The field of genetic science is very vast. Half the rate of first trimester abortions involves chromosomal abnormalities. Congenital malformations, childhood blindness, deafness, and mental retardation mostly occur due to gene mutations.

Chromosomes and disorders associated with the same:

Every cell in the human body constitutes chromosomes. The chromosome complement in humans consists of autosomes and sex chromosomes. There are 22 pairs of autosomes and a pair of sex chromosomes. They are known as X and Y chromosomes respectively. A normal male has 22 pairs of autosomes, an X, and a Y chromosome. A normal female has 22 pairs of autosomes and two X chromosomes. Karyotyping involves arranging the chromosomes as per the groups resulting in a photomicrograph. This photomicrograph or a karyotype consists of autosomes and sex chromosomes arranged in groups. Structural and numerical abnormalities in the chromosomes result in genetic disorders. They are known as chromosomal anomalies or chromosomal abnormalities.

Disorders due to an abnormal number of chromosomes are known as numerical anomalies. They arise due to non-disjunction of chromosomes. Monosomies (one chromosome less), trisomies (one chromosome extra), and many other conditions consist of chromosomal anomalies. Down’s syndrome is an example of trisomy. It occurs due to trisomy of the 21^st chromosome. Disorders associated with the structural abnormalities of chromosomes are known as structural anomalies. Deletions, duplications, inversions, and translocations arise due to the structural anomalies of the chromosomes. They also result in conditions such as mosaicism.

Disorders inherited due to faulty genes:

Monogenic or single gene disorders are the genetic disorders arising due to the inheritance of a mutated gene. Two main types of inheritance include autosomal inheritance and sex-linked inheritance. Autosomal inheritance involves autosome related disorders or traits. Inheritance of the traits due to the expression of genes present on the sex chromosomes is known as a sex-linked inheritance. Autosomal dominant inheritance manifests the trait even if the mutant gene occurs in a single dose. For example, Huntington’s disease arises due to abnormal CAG nucleotide repeat. Autosomal recessive inheritance manifests the trait even if the gene is present in the double dose (homozygous). An example includes sickle-cell anemia. Sex-linked inheritance is either X-linked or Y-linked. The X-linked inheritance occurs in a dominant or a recessive form. Very few cases report Y-linked inheritance.

Polygenic or multifactorial inheritance depends on many genes. The traits are known as quantitative traits and depend on many factors. Abnormalities in mitochondrial DNA (mtDNA) occur due to mitochondrial inheritance. The genes do not behave as dominant or recessive in polygenic inheritance. They exhibit an additive effect on the trait.

Biochemical genetics:

This area of genetics deals with the genetic control of the metabolic pathways. According to the one gene-one enzyme hypothesis proposed by Beadle and Tatum, metabolic processes occur in various steps controlled by enzymes. Each enzyme gets coded by one gene. Inborn error of metabolism arises due to enzyme unavailability or insufficiency occurring as a result of related gene mutations. The inborn error of metabolism follows Mendelian inheritance pattern. PKU, a classic example of the inborn error of metabolism, arises due to the deficiency of phenylalanine hydroxylase.

Image 2: The role of genetics in medicine

Detection of genetic disorders:

Cytogenetic and molecular genetic studies or tests detect various anomalies. The prenatal diagnosis helps to detect abnormalities in the fetus before birth. Examples of prenatal tests include Amniocentesis, chorionic villus sampling, fetoscopy, ultrasonography, maternal serum screening, and fetal blood sampling. Non-invasive prenatal tests also help in detecting the fetal DNA through maternal serum testing. Karyotyping helps to detect chromosomal abnormalities. FISH and other hybridization techniques detect minute changes in the DNA. SNP genotyping involves PCR, agarose gel electrophoresis, and gel documentation systems.

Genetic counseling:

It is a specialized session with a genetic counselor. The genetic counselor is an expert in genetics and a trained professional who guides the couples and patients suspected with the genetic disorders. Genetic counseling helps in the risk assessment of hereditary diseases, repeated abortions, and stillbirth cases. It also helps in reducing the risk of having a baby with the genetic and other ailments including the inborn errors of metabolism.

Gene cloning and DNA analysis:

Gene cloning involves cloning the gene of interest for incorporation into the desired vector. It has a wide range of applications in recombinant DNA technology. It helps in expressing the desired products such as proteins, vitamins, and other molecules. Peptide vaccines and hormones get developed using gene cloning. DNA analysis using techniques such as DNA fingerprinting help to solve parental issues. Analyzing DNA and genotyping techniques help in detecting gene mutations.

Gene therapy:

It helps in replacing the gene that has lost its function. The first step involves defective gene identification and cloning the normal gene in place of the defective gene. The normal gene insertion includes a vector. The first gene therapy gained success in case of a child having ADA deficiency associated with severe combined immunodeficiency syndrome (SCID). 

References:

[1] Emery’s elements of Medical Genetics, Peter D. Turnpenny

[2] Chromosome Abnormalities and Genetic Counseling,  R.J. MKinlay Gardner, Grant R Sutherland, Lisa G. Shaffer
[3] Medical Genetics, G. Bradley Schaefer, James N. Thompson

Copyright, 2018 All Rights Reserved

Paternity testing becomes simpler using DNA typing

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.

The working principle of DNA testing:

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 of DNA typing to determine paternity:

1.     Collection of blood samples:

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.

2.     Using restriction enzymes:

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.

3.     Separation of fragments using electrophoresis:

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.

4.     Southern blotting:

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.

5.     Probing:

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.

6.     Autoradiography

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.

7.     Data interpretation

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.

PCR Analysis:

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.

INFO-BOX

·        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. ·        The genome involves thirteen DNA regions possessing chances of variation ·        These studies involve scientists who conduct DNA profiling.

 Table: Information on genome and DNA typing

STR Analysis:

This analysis involves short tandem repeats. STR analysis uses highly polymorphic regions with short sequences.

Advantages of DNA paternity test:

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

                               

                                      © Copyright, 2018 All Rights Reserved.