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

                               

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Cloned gene analysis

Gene cloning or creating multiple copies of the desired gene and other sequences gets analyzed using various techniques. Cloning studies require information related to genes such as the position or the location of a gene on a chromosome, specific restriction sites, and their special arrangement. Cleaving the DNA with specific restriction enzymes help to target specific regions known as the restriction sites. A plethora of gene analyzing techniques involves restriction mapping, Southern blotting, northern blotting, and many others. Plasmid genomic libraries for specific DNA sequences or cloned DNA sequences get analyzed using DNA probes capable of hybridization. Labeling of the probes may or may not require radioactive labels. Techniques such as Southern or Northern blotting utilize probes. The probes have important properties. They help in recognizing complementary sequences in the nucleic acid molecule. It becomes simpler to identify and isolate specific DNA sequences from an organism. They play an important role in diagnostics and fingerprinting techniques.

The difference between the southern and northern blotting is simple. The Southern blot helps to blot the DNA. Northern blot helps in blotting RNA. Both the techniques involve electrophoresis for separating the nucleic acid fragments and probes for hybridization techniques. It is not possible to separate the DNA into fragments without digestion. The separation of fragments using electrophoresis requires a properly digested DNA. Hence, restriction mapping and blotting techniques go hand in hand.

Image: Cloned gene analysis

Restriction mapping:

A restriction mapping technique typically uses certain enzymes possessing the ability to cleave the DNA into fragments of particular sizes. The necessity of a restriction map is to locate the genes present on the chromosomes, study mutations associated with each polymorphism and lot more. Thus, restriction maps act like guides in cloning and molecular biology studies. A restriction enzyme cleaves the DNA at a particular site known as a restriction site. A nucleotide sequence specific to an endonuclease enzyme is known as a restriction site. For example, Eco RI, a restriction endonuclease, cleaves at guanine base of a 5’-GGATTC-3’ recognition sequence. The process of treating the DNA with the restriction enzyme is known as restriction digestion. The products obtained from the restriction digestion are known as restriction digests. A restriction digest consists of fragments of DNA of different sizes. In most of the restriction digestion experiments, the DNA sample gets treated with one or more restriction enzymes. The DNA samples get separately labeled. If one DNA sample gets treated with a particular restriction enzyme, the fragments obtained through this process get separated using electrophoresis. Suppose a DNA sample gets treatment with restriction enzyme  I whereas the other one gets treated with restriction enzyme II. 
Consider one more DNA sample treated with a combination of restriction enzyme I and II. After fragmenting the DNA samples, the next step involves loading the samples into the electrophoretic wells. Cleaving the DNA fragments with a single restriction enzyme reveals two bands. Cleaving a DNA sample with two restriction enzymes reveals three bands on the electrophoretic gel. The DNA fragments get separated based on their sizes. The smaller DNA fragments migrate faster. The larger DNA molecules migrate slowly. The comparison of the bands involves a marker DNA and control samples. The sizes of the fragments are noted down. A calibration curve construction involves distance migration on X-axis and Log Kb on the Y-axis. The interpretation of the results requires construct models. Restriction mapping is a kind of physical mapping technique. It helps in sequencing and analyzing the cloned DNA fragments. It is possible to find out whether a vector gets cloned properly using restriction mapping. Restriction digests get electrophoresed further for Southern blotting.

Southern blotting:

The electrophoresed gel consisting of DNA fragments get separated as per the sizes. Then it gets analyzed using hybridization and blotting procedures. The Southern blotting technique helps in blotting DNA bands. Discovered by E.M. Southern, this technique has a wide range of applications in cloning, SNP analysis, molecular testing, RFLPs, zoo blots, DNA fingerprinting, and microarray studies.

When DNA fragments get treated with the restriction enzymes and separated by gel electrophoresis, the bands of different sizes get separated on the gel. The next step involves placing the gel in a tray full of alkaline solution (buffer solution). The gel immersed in a buffer solution comes in contact with the buffer. The technique also utilizes a glass plate or a blotting paper. Next step involves covering the gel with a nitrocellulose filter paper. A stack of paper towels and weight gets placed on top of it. The blotting paper acts as a wick and carries the buffer solution from the tray to the gel, and finally to the membrane filter. The DNA fragments present on the gel come in contact with the buffer and get transferred to the membrane filter. Now, the membrane filter looks like a replica of the gel with distinct bands. The membrane gets further treated with the probe solution so that the hybridization takes place. Later on, the technique known as autoradiography helps in visualizing the bands. The Southern blotting technique helps to analyze cellular DNA for the presence of sequences complementary to the labeled probes. The cDNA molecule gets synthesized from a mRNA molecule and analyzed using Southern blotting.

Northern blotting:

It is a technique used to study RNA rather than DNA. It is similar to the Southern blotting technique. This technique reveals the size of the mRNA. It is used to investigate the presence of mRNA in a particular cell type or a tissue. The levels of gene activity get determined during the developmental stages. Once the electrophoretically separated RNA passes from the gel to an absorbent sheet, the RNA of interest gets revealed after hybridization. Transcription fo specific gene under certain environmental conditions gets detected using Northern blotting. Unregulation and downregulation of oncogenes and tumor suppressor genes can be studied. 

References:

[1] Recombinant DNA Technology, Sardul Singh Sandhu

[2] Recombinant DNA Principles and Methodologies, James Greene

[3] Gene Cloning and Manipulation, Christopher Howe

[4] Genetic Engineering, Verma P.S. & Agarwal V.K.

[5] Biotechnology-4: Including Recombinant DNA Technology, Environmental,  S. Mahesh
[6] Gene cloning and DNA analysis, T.A. Brown

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Nucleic acids

The nucleic acids are one of the six important biomolecules. They are also known as biopolymers. Friedrich Mischer discovered nucleic acids for the first time. Later on, various people found the role of nucleic acids in heredity and evolution. DNA, also known as Deoxyribonucleic acid, is mainly responsible for inheritance. DNA is not only found in chromosomes but also present in the mitochondria. It is known as the blueprint of life. A DNA molecule carries important instructions for the growth, reproduction and the development of an organism. Its structure looks like a double helix. RNA, also known as ribonucleic acid, is mainly found in the nucleolus. It is also present in the cytoplasm and the ribosomes. RNA is a single-stranded structure. It codes for a protein in a common cellular process known as translation. Nucleotides arranged in the RNA are linear. The nucleotides are small building blocks of DNA and RNA. They are the basic structural units of DNA and RNA.

The genetic material:

A wide storage of an individual’s information related to the characteristic traits is known as the genetic material. The cells are the storage houses of the genetic material. It possesses an ability to get transferred from the parent to the progeny. The genetic material, mainly consisting of DNA, is present in most of the organisms. Certain viruses possess only RNA as their genetic material. There are differences among the genetic materials of the prokaryotes and the eukaryotes. Very few chromosomes are present in the prokaryotes. In most of the prokaryotes, the cells contain double-stranded DNA. The eukaryotic cells have many chromosomes. The DNA is compact. Just like folding a long thread into several small folds, the DNA fits into the chromosomes. The genes are parts and parcels of the DNA. They are involved in protein-coding and other activities. The DNA looks like a long chain. Unlike DNA, RNA is not responsible for inheritance. Both the nucleic acids are known as polymers. A polymer is a large molecule consisting of many similar small molecules with specific linkage. These similar molecules are known as monomers. The scientists studied different aspects of the genetic material and structured various experiments.

Image 1: The genetic material

Structure of the nucleic acids:

The DNA and RNA polymers consist of monomers known as nucleotides. A nucleotide contains three main parts such as a pentose sugar, nitrogenous base, and a phosphate bond. The nucleotides are linked together by covalent linkages. These bonds form linkages between phosphate group of one nucleotide and the third carbon of the sugar present on the other nucleotide.  The 5’-3’ phosphate linkages are known as phosphodiester bonds. They are strong stabilizers of the nucleic acid backbone. The DNA consists of a pentose sugar known as deoxyribose. RNA has a ribose sugar.

There are two classes of nitrogenous bases. They are known as purines and pyrimidines respectively. Purines are nine-membered structures. They have double rings. Pyrimidines are six-membered structures. They are single ringed. Examples of purines are adenine and guanine. Examples of pyrimidines are thymine, cytosine, and uracil. 

The chemical composition of DNA:

A DNA consists of three important chemical compounds as follows:

1.     Sugar: As discussed earlier, the DNA consists of a deoxyribose sugar. It is a pentose sugar. It consists of five carbon atoms. A deoxyribose sugar is a monosaccharide. Loss of an oxygen atom from the ribose sugar gives rise to deoxyribose sugar. An enzymatic reaction involving ribonucleotide reductase synthesizes the sugar.

2.     Nitrogenous base: They consist of one or more nitrogen atoms. There are four types of nitrogenous bases in a DNA mainly adenine, guanine, thymine, and cytosine. A nitrogenous base bonds two nucleotides. These bases form hydrogen bonds between the two DNA strands and look like a twisted ladder. Adenine pairs with thymine and Cytosine pairs with Guanine.

3.     Phosphoric acid or a phosphate group: The sugar and the base combine to form a nucleoside. A phosphate group gets added to a nucleoside and results in the formation of a nucleoside phosphate. A nucleotide is also known as nucleoside phosphate. The 5’-group of the sugar is attached to the phosphate group. There is a covalent bond between the phosphate group of one nucleotide and 3’ carbon of the sugar present on the other nucleotide. The phosphodiester bonds are very strong. 

Image 2: Nucleic acids

The molecular structure of the DNA:

The double helical structure consists of nitrogenous bases paired with hydrogen bonds for maintaining the complementary structure. The adenine base pairs with the thymine using two hydrogen bonds. The guanine and cytosine pair using three hydrogen bonds. The helix involves specific turn, measured in nanometers. Each turn measures 3.4 nanometres giving a diameter of 0.34 nanometers for the double helix.

Watson and Crick published their works in 1953. Their double helix model of DNA had the features describing the structure and orientation of the double helix. Watson and Crick deduced that the DNA molecule consisted of two polynucleotides wound around each other. Their orientation was clockwise or right-handed. The strands were anti-parallel to each other. The orientation of the bases is toward the central axis. The hydrogen bonds between the bases help the strands to get separated easily. They also found an unequal spacing between the two sugar-phosphate backbones. The unequal spacing results into two types of grooves. Major groove is wide. The narrow groove is known as a minor groove. The nitrogenous bases can make contacts with the proteins for further packing of DNA into structures known as chromosomes.

 Structure of RNA

The nucleolus and the cytoplasm are the places where the RNA resides. DNA synthesizes RNA. This process is known as transcription. Just like DNA, an RNA molecule consists of the three main chemical components.

a.     A ribose sugar is present in the RNA. It is a five-carbon sugar. It is mainly involved in generating the energy molecule known as ATP.

b.    An RNA molecule consists of Adenine, Guanine, Cytosine, and Uracil.

c.      The third component includes the phosphate groups.

The messenger RNA or the mRNA gets synthesized from the DNA inside the nucleus. It consists of both introns and the exons. After splicing, the RNA gets matured to form a mature mRNA consisting of only the exons. The process of translation or protein synthesis occurs outside the nucleus. The ribosomal RNA or the rRNA occurs in the ribosomes. These components play a crucial role in translation. The rRNA plays a crucial role in the organization of the nucleolus. The transfer RNA (tRNA) is an important component of translation. 

References:

[1] Principles of Nucleic Acid Structure, Stephen Neidle, 2010, Preview
[2] Medical Genetics, G.P. Pal, first edition

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What is genetics?

Introduction to Genetics:

The subject known as genetics involves the study of genes, their mechanisms, regulation, and their pattern of expression. We must know about the genes to study the subject. Specific sequences of the nucleotides are known as the genes. They express a particular phenotype. These entities are also known as the functional units of heredity (transmission of genes from one generation to the next). We all resemble our parents, yet look different. The physical, mental and behavioral characteristics get passed on from the parents to the children, through gametes. In eukaryotes, the genetic material is present in a membrane-bound nucleus within the cells.

Differences between organisms are due to differences in their genetic makeup. These differences occurred due to events such as mutation, recombination, and selection. Mutation leads to a change in the genetic material. The exchange of genetic material between chromosomes is known as recombination. A particular combination of genes chosen in a given environment is known as selection. Mutations may either occur spontaneously or occur due to mutagens. They get permanently incorporated in the genetic code.

Image 1: Introduction to genetics

Basic Terms in Genetics

We must know the basic terms used in genetics.

Cells: The basic functional units in the body, with a full-fledged genetic and biochemical, machinery, are known as cells. They synthesize certain molecules necessary for the body, carry out regulation mechanisms, participate in signaling, and undergo a cascade of events. Right from the synthesis of energy to the functioning of every body part requires the pre-planned efforts of the cells.

Nucleus: Inside the cell, there is a nucleus. It is the main store of genetic machinery.

Chromosomes: The thread-like structures present in the nucleus are known as chromosomes. The chromosomes have different shapes.

Base Pairs: There are four bases in DNA such as Adenine, Guanine, Thymine, and Cytosine. In RNA, uracil is an alternative to the thymine. The sequence of these bases within the strand determines the genetic information.

DNA: Deoxyribonucleic acid or DNA is the hereditary material present in the nucleus of the cell. It is also known as a blueprint for life. It is in the form of a double helix with base pairs attached to the sugar-phosphate backbone.

RNA: The ribonucleic acid is involved in protein synthesis.

In more sophisticated terms, a gene is a sequence that codes for a functional molecule. Gene expression is an important process in all the organisms.

Phenotypic traits: These are the specific characteristics passed on from the parent to offspring. Examples include eye color, skin type, the shape of the nose, etc.

Genotype: Different DNA sequences constitute a genotype. Thus, genotypes combine with the environmental factors and determine the trait or phenotype.

Homozygous organism: An organism having a pair of identical alleles is said to be homozygous.
Heterozygous organism: An organism having two different alleles is said to be heterozygous.

The Discovery of Genetics

Mendelian Genetics (1856-1866):

Gregor Mendel was an Austrian monk in the 19th century. He was the pioneer in genetics. His experiments on pea plant led to the discovery of the basic mechanisms of the heredity. Mendel established the basic principles of heredity. This study is known as Mendelian genetics. Later on, various researchers came up with interesting concepts in the field of genetics.

Gregor Mendel is known as the father of modern genetics. He worked on pea plants.

Image 2: Mendel and his study on the pea plants

The DNA era (1944-1972):

It started with Avery's experiment which demonstrated isolation of DNA as a genetic material (also known as transforming principle). Transposons were discovered later on by other scientists.

Discovery of Genomic science (1972- 2016):

The genomics era started with Walter Fiers's discovery of the genetic sequence for a bacteriophage. Likewise, Sanger, Maxam, and Gilbert sequenced the DNA for the first time. The computer stores important information in databases. There are many genetic databases available on the internet. One of the examples of genetic databases includes the National Center for Biotechnology Information (NCBI). A genetic database consists of a store of researched documents, computational biology data, genomic data software and other useful information. Gene maps show the location of genes on chromosomes. The position of a gene on the chromosome is known as gene locus. The unit of genetic distance is known as the map unit. Genetic maps are used to study the organization of genes on the chromosomes. They are used to obtain complete genome sequences.

Many discoveries led to advances in genomic science such as:

·        The DNA sequencing

·        Nucleic acid labeling

·        Mapping the structure of DNA

·        Gene Cloning

·        Transposon-mediated mutation and chromosome breakage studies

·        DNA fingerprinting

·        CFTR protein sequencing

·        Identification of BRCA gene

·        Cloning of Dolly sheep

·        Genome sequencing of Drosophila

·        Development of Human Genome Project

·        Genetic Databases and Maps

Branches of Genetics

Four main branches of genetics are:

Human genetics: It is the study of inheritance in humans.

Plant genetics: It is the study of genes and inheritance in plants.

Animal genetics: It involves the study of genes in animals. Genetic engineering is used to breed animals with a specific trait. Animal geneticists develop genetically modified animals.

Microbial genetics: Various genetic mechanisms also occur in microbes such as bacteria, fungi, and viruses. Microbial genetics is the study of genetic mechanisms in microbes.

Image 3: Branches of genetics

Based on the above categories, genetics can be further studied as follows:

Classical genetics: It is the oldest branch of genetics that depends only on the physical characteristics of an organism. It involves the study of Mendelian Inheritance.

Molecular genetics: It involves the study of the structure and function of the genes at molecular levels.

Cytogenetics: It mainly deals with chromosomes and cellular behavior.

Biochemical genetics: It involves the study of biochemical processes, metabolic disorders and the role of genetic machinery.

Medical genetics: It helps in diagnosing genetic disorders.

Epigenetics: It is the study of heritable changes in gene function under the influence of certain environmental factors. It covers important aspects such as DNA methylation and histone modification.

Developmental genetics: It is the study of genes and their way of controlling the growth and development of an organism.

Behavioral genetics: It involves genetic science to understand the behavior of an individual or an organism.

Population genetics: It is a study based on evolutionary biology and Mendelian inheritance. Population genetics involves the study of particular traits, changes in alleles and genotypes in a population.

Ecological genetics: It involves a combination of genetics and ecological sciences. In layman terms, ecological genetics is the study of natural populations.

Genetic engineering: It is a direct way of manipulating the gene of an organism to get the desired trait.

Genetics of intelligence: It uses genetics to determine the I.Q. of an individual.

Genomics: It is a study of structure, function, evolution, and mapping of the genomes.

Importance of Genetics in Medicine

A large number of diseases have a genetic background. That is why genetics plays a crucial role in medical science. Genetics may be used to avoid disorders related to genes and chromosomes. With the latest genetic techniques such as prenatal diagnosis, it is easy to detect the chances of abnormalities in the fetus.

   

Role of Medical Genetics

Diagnosing genetic disorders Assessing the risk of occurrence of the disease Genetic counseling Avoiding repeated spontaneous abortions Assisting in cousins marriages and disputed paternity Management of genetic disorders Pharmacogenomics and personalized medicine Deciding dietary patterns

Table: Role of Genetics in medicine and healthcare

Genetic Engineering

Genetic engineering involves modification or cloning a specific gene preferably involving a vector. Many drugs and hormones such as insulin, somatostatin, blood clotting factors and growth hormones are synthesized using recombinant DNA technology. Synthetic vaccines such as anti-rabies, anti-malaria, anti-hepatitis vaccines are produced using genetic engineering.

Genetic alterations to the plants may improve the quality of the crop. Such crops may be able to satisfy human needs. For example, disease resistance to plants can be achieved using gene alteration. Other desirable traits include stress resistance, drought resistance and tolerance to extreme conditions, salinity and temperatures.

References:

[1] Genetics, Daniel Hartl, 2011, Preview
[2] IGenetics, a molecular approach, Peter Russel, second edition

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