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.

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

                                       © Copyright, 2018 All Rights Reserved.

A Review on Restriction Enzymes

The enzymes involved in cleaving the specific regions in the genome are known as restriction enzymes. They are also known as molecular scissors. They are used widely in molecular biology. A restriction site is a specific region in the DNA consisting of bases cleaved by these enzymes. The process in which the restriction enzyme cleaves at a particular site is known as restriction digestion. The products in the form of the nucleic acid fragments are known as the restriction digests. They are known to play a crucial role in mapping, sequencing, and cloning. Most of the restriction enzymes belong to the class of endonucleases. It is an enzyme cleaving or hydrolyzing the phosphodiester bond within a DNA. The knife and fork model of DNA replication represents a triangular structure. This triangular structure is a sign of an endonuclease enzyme. The DNA molecule consists of internal phosphodiester bonds which get cleaved through restriction endonucleases.

In somatic tissue or cells, these enzymes are capable of hydrolyzing DNA by introducing double-strand breaks. Naturally occurring restriction endonucleases are known as the cell protectors. They protect the cells from viral infections. For research and analysis purpose, the extraction source of the enzymes involves microorganism or chemical synthesis.

Properties of restriction endonucleases:

A restriction enzyme recognizes a restriction site at 3’ carbon and phosphate group of a phosphodiester bond. The resultant fragments have 5’ phosphate group and 3’ hydroxyl group. Bacteria and green algae chlorella have naturally occurring restriction enzymes. Bacteria undergo a process of methylation and modify the restriction sites. Hence, the bacterial cells get protected from the action of restriction enzymes. Arbor, Nathans, and Smith received a Nobel prize in discovering the restriction enzymes. Many restrictions enzymes select sites having an axis of symmetry. The number of cuts made by restriction enzymes gets determined by the frequency of occurrence of the restriction sites. The restriction sites occur in a randomly distributed base pair. The formula (1/4)ⁿ indicates the probability of the occurrence of the restriction site with a 50% GC content. (Where n indicates the no. of nucleotide pairs in the recognition sequence).

Image: Restriction digestion (Treatment of the DNA sample with restriction endonucleases leads to the synthesis of fragments of different sizes.

Nomenclature:

The nomenclature of the restriction enzymes involves the organisms from which they were isolated. The first letter belongs to the genus and the second and third letter from the species. It involves an italic or underlined font followed by Roman numerals. For example, EcoRI is an example of a restriction enzyme obtained from E. coli strain RY13. Thus, ‘Eco’ belongs to E. coli, and RI belongs to the strain RY13. There are four classes of the restriction enzymes based on the recognition sequences.

Enzymes with no. of recognition sequences in base pairs	Property
4	Symmetrical sequences
6	Symmetrical sequences
8	Symmetrical sequences
spacer sequence	Non-specific sequences

Table: Enzymes and their properties 

Constructing a restriction map:

A restriction map is a kind of a physical map. It consists of a piece of DNA with restriction sites specific for endonucleases. The number of bases decides the sites of separation. Restriction enzymes create two types of ends such as sticky or blunt ends. A staggered cut in the restriction site with symmetrical nucleotide sequence generates sticky ends. There are two types of ends such as 5’ or 3’ overhanging ends. When a restriction enzyme cuts at a symmetrical nucleotide sequence of a restriction site between the two base pairs, the blunt ends get created. When the DNA gets digested with the restriction enzymes, and the resultant fragments get separated on a gel, the banding patterns are visible after staining. They are known as restriction fragment length polymorphisms (RFLPs). Sometimes the restriction sites vary in different individuals. A restriction enzyme cleavage site present in an individual may be absent in another individual. RFLPs help in mapping the genes or polymorphic sites. However, the non-polymorphic sites get missed. Restriction mapping solves this problem. A single restriction enzyme or combination of restriction enzymes cleave the DNA efficiently. Thus we obtain the fragments of different sizes measured in kilobases. The electrophoretic gel separates the fragments based on their sizes. Autoradiography helps to visualize the bands and cut the desired portion of the gel to take the DNA fragment.

Cloning Vectors:

A cloning procedure primarily uses a piece or a fragment of DNA obtained from the restriction digestion. The fragment of DNA gets inserted into the vector. The technique involves two main steps. First, the plasmid gets cleaved at a unique restriction site with an appropriate restriction enzyme. The second step involves the insertion of a piece of DNA cut with the same enzyme. The procedure further involves mixing of the cloning vector and a DNA fragment. It results into base pairing and annealing the two single-stranded ends. An enzyme known as DNA ligase acts as a sealing agent. It seals the gaps formed due to a phosphodiester bond.

Applications of restriction enzymes:

1.     DNA typing:

DNA fingerprinting or DNA typing determines the paternity and maternity of an individual. It helps in crime investigation. The establishment of variability in the ethnic groups involves DNA typing. Endangered species determination and genetic variability studies involve DNA typing techniques. Forensic medicine largely involves DNA fingerprinting techniques. Initially, the DNA obtained from the suspect individual gets a treatment with the restriction enzymes. The restriction digests obtained from the procedure include DNA fragments of different sizes. The fragments get separated as per their sizes in an electrophoretic apparatus. Using a Southern blot and a probe hybridization technique, visualization of the DNA bands of the suspect involves autoradiography.

It is not possible to separate the DNA fragments without the restriction enzymes. Hence, obtaining the information of the suspect invites difficulties. In the detection of paternity or maternity cases, the DNA obtained from the child and his alleged parents have to undergo restriction enzyme treatment. Autoradiogram shows a banding pattern of each sample as a DNA fingerprint.

2.     DNA molecular testing:

Genetic testing such as disease gene detection requires restriction enzyme treatment. The requirement of the restriction enzymes using PCR determines the presence of microsatellites. RFLP analysis and restriction mapping utilize the restriction enzymes. Detection of genes associated with diseases such as sickle cell anemia, phenylketonuria (PKU), and others become easy to detect.

3.     Gene Cloning:

The isolated DNA gets cleaved through RE treatment. The fragments obtained through RE treatment mix with the cloning vector and get integrated into the restriction sites in the cloning vectors. The cloning vector gets transformed into the host and allowed to replicate. The process is known as molecular cloning. Identical copies of DNA of interest obtained from the above process are known as clones. Expression of genes, synthesis of protein products, gene therapy, and many other important applications require gene cloning.

4.     Preparing recombinant vectors:

Restriction enzyme treatment targets multiple cloning sites or the restriction sites in a plasmid or any other vector. The fragment of interest gets inserted into the multiple cloning sites or a polylinker and gets transformed into the host. The host (bacterial cells) plated on a suitable medium show the growth of the recombinant colonies.

5.     Industrial applications:

It is possible to grow plant hybrids showing resistance to herbicides, pesticides, and drought tolerant varieties. Restriction digests obtained through restriction enzyme treatment get inserted into a vector. Then the DNA gets microinjected into the animal’s pronucleus to obtain transgenic animals. Hence, it is possible to produce a recombinant protein product using a transgenic animal. Example, the gene of interest expressed in a mammary tissue helps protein secretion in the milk.

6.     Genomic and cDNA libraries:

 The recombinant DNA libraries consist of a collection of clones with the desired DNA sequence. The fragments subjected to restriction enzymes get cloned into a vector to obtain a collection of clones known as genomic or cDNA libraries. 

References:
[1] Essential Genetics: A Genomics Perspective, Hartl, Elizabeth W. Jones
[2] Molecular Biology, David P. Clark, Nanette J. Pazdernik
[3] Genetic Engineering, Verma P.S. & Agarwal V.K.
[4] Molecular Biology and Genomics, Cornel Mulhardt

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