Practical Application of DNA Fingerprinting for Forensic Veterinary Medicine
DNA Fingerprinting
DNA fingerprinting (also called DNA profiling, DNA testing, or DNA typing) is a forensic technique used to identify individuals by characteristics of their DNA. A DNA profile is a small set of DNA variations that is very likely to be different in all unrelated individuals, thereby being as unique to individuals as are fingerprints (hence the name for the technique).
Although 99.9% of human DNA sequences are the same in every person, enough of the DNA is different that it is possible to distinguish one individual from another, unless they are monozygotic (“identical”) twins. DNA fingerprinting uses repetitive sequences that are highly variable, called variable number tandem repeats (VNTRs). Modern law enforcement in particular uses short tandem repeats (STRs). STR loci are very similar between closely related individuals, but are so variable that unrelated individuals are extremely unlikely to have the same STRs. The combination of STRs used by law enforcement enable identification though because even closely related individuals will not share all the same STR loci.
The modern process of DNA fingerprinting was developed in 1984 by Sir Alec Jeffreys, while he was working in the Department of Genetics at the University of Leicester. DNA fingerprinting can be used to identify a person or to place a person at a crime scene and to help clarify paternity. DNA fingerprinting has also been widely used in the study of animal and floral populations and has revolutionized the fields of zoology, botany, and agriculture.
DNA fingerprinting is a method used to identify living things based on samples of their DNA. Instead of looking at the whole sequence of a person’s DNA, these techniques look at the presence or absence of common markers that can be quickly and easily identified.
It is almost impossible for a person to commit a crime without leaving behind a trace of his or her DNA. Hair, stains of blood, and even conventional fingerprints contain traces of DNA. The sensitivity and evidential power of DNA Fingerprinting have impacted the way crime scenes are being investigated. DNA fingerprinting also called DNA typing, DNA profiling, genetic fingerprinting, genotyping, or identity testing. The aim of using genetic analysis for forensic casework is to produce a DNA profile that is unique to each individual. DNA fingerprinting offers forensic examiners remarkable opportunities to obtain and present to the court highly relevant evidence from semen specimens in relation to sexual assaults. It is of great use both to forensic investigators and to legal counsel. It has the ability to match a suspect to body fluids found at the scene of the crime. This article points out the practical applications of forensic DNA fingerprinting.
DNA is also known as Deoxyribo Nucleic acid, is a chemical structure that includes a specific sequence of bases called nucleotides which contain the information of all the characteristics of living organisms. DNA has a double helix structure. It is made up of two helical chains or strands. These strands are spirally coiled around a central axis, all along their length. The four nucleotides that make up the sequence of DNA are adenine (A) guanine (G), cytosine (C) and thymine (T). The structure of DNA is same in all individuals, but the only differentiating point is the order of base pairs. The presence of millions of base pairs in an individual’s DNA creates a different sequence that is unique from each other. In the process of identification forensic scientists scan certain regions of DNA that vary from each other and utilizes the data to create a DNA profile of that person. DNA profiling is also known as DNA fingerprinting which is an important forensic technique to identify the suspects and the victims.
History:
DNA Fingerprinting is a method which is used to identify individuals on the basis of the molecular characteristics of the DNA. The procedure of DNA fingerprinting, involves comparing samples of human DNA left at a crime scene with DNA obtained from a suspect, which is now considered as the most authentic form of identification by many investigators and scientists. In 1980, Wyman and White discovered the first DNA fingerprinting technique called Restriction fragment length polymorphism (RFLP). In 1985, Jeffrey’s described the first development of multilocus DNA fingerprints and considered that these individual-specific DNA patterns may provide a powerful method for identification of individuals and paternity testing. In 1987, Nakamura coined the term variable number of tandem repeats (VNTR) to describe individual loci where alleles are composed of tandem repeats that vary in the number of core units. In 1985, Kary Mullis developed a technique called polymeric chain reaction (PCR) into forensic work. By allowing the selective amplification of any desired stretch of DNA, PCR helped in low- level DNA detection at scene of crime. Now-a-days most of the forensic genetic methods are based on PCR.
DNA fingerprinting has become standard method in forensic genetics especially in criminal forensic casework such as stain analysis, DNA analysis of hair, individual identification and paternity testing. The potential of DNA fingerprinting has also made possible the resolution of immigration problems. DNA fingerprinting plays a major role in rapid identification of individuals in mass disaster cases. The techniques of DNA fingerprinting aids in the investigation of sexual assault and rape cases
The process is sometimes called “DNA testing” or “DNA profiling”, but signals the same process. Early DNA fingerprinting was developed in the years before the whole human genome had been sequenced. DNA fingerprinting typically relies on short tandem repeats (STRs), which are unique to individuals. These sections of DNA can be compared between two different samples. If they show the same pattern after gel electrophoresis, it indicates that the samples are from the same source.
A DNA fingerprint looks something like the columns on the paper below. On this paper, each dark band represents a fragment of VNTRs – and each column is a different tissue sample. A match would be indicated by two columns whose VNTRs patterns matched precisely.
DNA profiling is different from genetic testing, in which a DNA sample is tested to see if it contains genes for inherited diseases or other traits. The FBI and other law enforcement agencies use the CODIS index, which compares 13 sections of DNA and can accurately identify criminals based on a DNA sample.
DNA Fingerprinting Uses
This process is frequently used in criminal investigations to determine whether blood or tissue samples found at crime scenes could belong to a given suspect. This technology is also used in paternity tests, where comparison of DNA markers can show whether a child could have inherited their markers from the suspected father.
In science, DNA fingerprinting is used in the story of plant and animal populations to determine how closely related species and populations are to other species and populations. Further, it can track their spread over time. This ability to look directly at an organism’s gene markers has revolutionized our understanding of zoology, botany, agriculture, and even human history.
DNA Fingerprinting Steps
Extracting DNA from Cells
To perform DNA fingerprinting, you must first have a DNA sample! In order to procure this, a sample containing genetic material must be treated with different chemicals. Common sample types used today include blood and cheek swabs.
These samples must be treated with a series of chemicals to break open cell membranes, expose the DNA sample, and remove unwanted components – such as lipids and proteins – until relatively pure DNA emerges.
PCR Amplification (Optional)
If the amount of DNA in a sample is small, scientists may wish to perform PCR – Polymerase Chain Reaction – amplification of the sample.
PCR is an ingenious technology which essentially mimics the process of DNA replication carried out by cells. Nucleotides and DNA polymerase enzymes are added, along with “primer” pieces of DNA which will bind to the sample DNA and give the polymerases a starting point.
PCR “cycles” can be repeated until the sample DNA has been copied many times in the lab if necessary.
Treatment with Restriction Enzymes
The best markers for use in quick and easy DNA profiling are those which can be reliably identified using common restriction enzymes, but which vary greatly between individuals.
For this purpose, scientists use repeat sequences – portions of DNA that have the same sequence so they can be identified by the same restriction enzymes, but which repeat a different number of times in different people. Types of repeats used in DNA profiling include Variable Number Tandem Repeats (VNTRs), especially short tandem repeats (STRs), which are also referred to by scientists as “microsatellites” or “minisatellites.”
Once sufficient DNA has been isolated and amplified, if necessary, it must be cut with restriction enzymes to isolate the VNTRs. Restriction enzymes are enzymes that attach to specific DNA sequences and create breaks in the DNA strands.
In genetic engineering, DNA is cut up with restriction enzymes and then “sewn” back together by ligases to create new, recombinant DNA sequences. In DNA profiling, however, only the cutting part is needed. Once the DNA has been cut to isolate the VNTRs, it’s time to run the resulting DNA fragments on a gel to see how long they are!
Gel Electrophoresis
Gel electrophoresis is a brilliant technology that separates molecules by size. The “gel” in question is a material that molecules can pass through, but only at a slow speed.
Just as air resistance slows a big truck more than it does a motorcycle, the resistance offered by the electrophoresis gel slows large molecules down more than small ones. The effect of the gel is so precise that scientists can tell exactly how big a molecule is by seeing how far it moves within a given gel in a set amount of time.
In this case, measuring the size of the DNA fragments from the sample that has been treated with a restriction enzyme will tell scientists how many copies of each VTNR repeat the sample DNA contains.
It’s called “electrophoresis” because, to make the molecules move through the gel, an electrical current is applied. Because the sugar-phosphate backbone of the DNA has a negative electrical charge, the electrical current tugs the DNA along with it through the gel.
By looking at how many DNA fragments the restriction enzymes produced and the sizes of these fragments, the scientists can “fingerprint” the DNA donor.
Transfer onto Southern Blot
Now that the DNA fragments have been separated by size, they must be transferred to a medium where scientists can “read” and record the results of the electrophoresis.
To do this, scientists treat the gel with a weak acid, which breaks up the DNA fragments into individual nucleic acids that will more easily rub off onto paper. They then “blot” the DNA fragments onto nitrocellulose paper, which fixes them in place.
Treatment with Radioactive Probe
Now that the DNA is fixed onto the blotting paper, it is treated with a special probe chemical that sticks to the desired DNA fragments. This chemical is radioactive, which means that it will create a visible record when exposed to X-ray paper.
This method of blotting DNA fragments onto nitrocellulose paper and then treating it with a radioactive probe was discovered by a scientist name Ed Southern – hence the name “Southern blot.”
Amusingly, the fact that the Southern blot is named after a scientist and not the direction “south” did not stop scientists from naming similar methods “northern” and “western” blots in honor of the Southern blot.
X-Ray Film Exposure
The last step of the process is to turn the information from the DNA fragments into a visible record. This is done by exposing the blotting paper, with its radioactive DNA bands, to X-ray film.
X-ray film is “developed” by radiation, just like camera film is developed by visible light, resulting in a visual record of the pattern produced by the person’s DNA “fingerprint.”
To ensure a clear imprint, scientists often leave the X-ray film exposed to the weakly radioactive Southern blot paper for a day or more.
Once the image has been developed and fixed to prevent further light exposure from changing the image, this “fingerprint” can be used to determine if two DNA samples are the same or similar!
DNA as a Forensic Tool
Information and clues obtained from DNA samples found at crime scenes have been used as evidence in court cases, and genetic markers have been used in forensic analysis. Genomic analysis has also become useful in this field. In 2001, the first use of genomics in forensics was published. It was a collaborative attempt between academic research institutions and the FBI to solve the mysterious cases of anthrax communicated via the US Postal Service. Using microbial genomics, researchers determined that a specific strain of anthrax was used in all the mailings.
Mitochondrial Genomics
Mitochondria are intracellular organelles that contain their own DNA. Mitochondrial DNA mutates at a rapid rate and is often used to study evolutionary relationships. Another feature that makes studying the mitochondrial genome interesting is that the mitochondrial DNA in most multicellular organisms is passed on only from the mother during the process of fertilization. For this reason, mitochondrial genomics is often used to trace genealogy.
Applications of DNA Fingerprinting in wildlife crimes:
Advances in DNA technology in the recent years have altered the investigation of many offences. The utilization of these techniques also has direct application to many wildlife related offences. Examination of non-human DNA can provide a vital proof in wildlife crime investigations. DNA is present in a different types of evidence ranging from blood, bone and feathers to powdered plant material, animal saliva and other transferable traces. After the development of the first human DNA fingerprinting method by Sir Alec Jeffrey’s it was quickly realized that parallel techniques could be applied for other species; such as birds. This led to forensic genetic evidence being used to support a successful prosecution relating to the theft of wild hawks in the UK.
DNA profiling is currently applied to link trace evidence items to victims of wildlife crime in cases of poaching, theft and animal persecution across a wide range of species. Also used to verify family relationships as a part of investigations into the laundering of wild animals through captive breeding programmes. Additionally to the individual identification; DNA profiling techniques may be used to match a sample to its geographic origin. This has numerous possible applications from investigating the illegal timber trade to enforcing fishing regulations.
In sexual offences:
Earlier to the use of DNA fingerprinting, the cases of sexual assaults solved only by the use of circumstantial evidences. In sexual offences as there will be no eyewitness it will be difficult for the victim to prove the offence. The discovery of DNA created a great hope in dealing the cases of sexual offences. Biological evidentiary clues such as semen, saliva and swabs are important in DNA fingerprinting and aids in the identification of suspect. For instance, saliva is found in a variety of offences like murders, scuffles, sexual offences specifically in bitemarks of victim or suspect, on cigarette butts, tea cups etc. usually carry saliva stains. DNA fingerprinting from the semen, swabs and saliva found at the scene of crime helps in arresting the suspects rapidly. The contamination of semen with other body materials such as vaginal fluids, rectal matter in sodomy cases, saliva in cases of oral sex or semen of multiple rapists in gang rapes every culprit can be individualized and identified by the process of DNA fingerprinting.
In Identification of Individuals from skeletal remains:
The chances of analysis of skeletal remains has been of great interest in the field of forensic science, because such remains are frequently preserved for very long periods of time and in cases like mass disasters they are the only source of information available for analysis. The preparation of DNA profiles from skeletal remains can be essential in the personal identification of missing persons and victims of mass disaster cases. The generation of DNA profile from bones and teeth plays an important role in the procedure of identification of unidentified remains cases.
In Disaster victim identification:
Analysis of DNA is one of the most important technique in identification of mass disaster victims. DNA fingerprinting is used to identify victims with their mutilated and fragmented body parts, and also used to identify criminals. DNA fingerprinting used for the first time in disaster victim identification is in the year 1990 i.e., in identification of victims of fire on Scandinavian Star ferry. The identification of victims through DNA analysis is by the collection of ante-mortem and post-mortem samples. Ante-mortem samples such as any personal objects used by victims, buccal swabs, and pathology specimens. Post-mortem samples includes the DNA samples obtained from bones such as femur and metatarsal bones, teeth specifically molar teeth, muscle tissues are also used if body is not in the stage of decomposition. DNA analysis of victims is also done by using Y-STR typing, mtDNA, autosomal STR markers, and Amelogenin markers for the identification of sex of the victims. SNP’s are very useful in the conditions where DNA are samples are degraded [7]. Commercially available kits such as AmpFLSTRw Profiler Plusw ID and COfilerw PCR Amplification Kits, AmpFLSTRw Identifilerw PCR Amplification Kit, the AmpFLSTRw SGMw Plus PCR Amplification Kit (Applied Biosystems, Foster City, CA), and the PowerPlexw 16 System (Promega Corp., Madison, WI) aids with STR typing of mass disaster samples.
In Differentiating Monozygotic Twins:
Differentiation of monozygotic twins is not possible because they have identical autosomal STR profiles. But through recent discovery of extremely rare mutations by ultra-deep next-generation sequencing (NGS) the individualization or differentiation of monozygotic twins is possible. Paternity disputes of monozygotic twins are rare. This technique is very useful if such cases are recorded.
In kinship analysis:
DNA fingerprinting can also be used to identify whether the two or more individuals are members of the same family. This type of analysis is also known as kinship analysis, which is applied frequently in paternity testing. DNA fingerprinting is capable of resolving the uncertainty about a child’s paternity prenatally. This can be of major significance to sexual assault victims, women with multiple sexual partners, and husbands suspicious of their wives adultery. Autosomal STR markers are mostly used in the study of forensic case works. Mitochondrial DNA can be used to examine the maternal line and Y-STRs are used in determining the paternal-line inheritance patterns.
In determination of sex:
DNA fingerprinting can also be used to identify the sex of an individual. Forensic scientist’s deals with the bodies that are so badly damaged that analyzing DNA might be the only way of identifying their sex. The genetic difference between the sexes is the possession of a Y chromosome by males, so detection of DNA specific for Y chromosome aids in differentiating between males and females. A PCR directed at Y chromosome specific DNA sequences would give a product with male DNA but no bands if the sample comes from a female. Another technique such as PCR of the amelogenin gene which codes for the protein found in tooth enamel. But both the techniques have certain limitations and are no longer necessary.
In identification of missing persons:
DNA fingerprinting has extreme importance in identifying missing persons when compared to other forensic techniques of human identification such as dactyloscopy, Forensic odontology and anthropology. Because DNA can be extracted from any tissue i.e., soft tissue, bones, teeth and even from degraded tissues. Countries like Spain and US implemented certain programs to identify the missing individuals by using DNA fingerprinting. The Spanish Phoenix Program (Programa FENIX) includes two databases that contains STR profiles and mtDNA sequences of questioned samples and reference samples from relatives. This program analyze DNA and establishes a link between the reference samples from relatives of missing persons and questioned samples such as unidentified bodies or skeletal remains of previously unsolved cases. In US, FBI (Federal bureau of investigation) helps by using NMPDD (National Missing Person DNA Database) Program. The US database contains three indexes such as biological relatives of missing persons, unidentified human remains and missing persons in which DNA profiles can be entered. The method involves in this program is DNA fingerprinting of genetic markers such as autosomal STRs, Y-chromosome STRs and mtDNA.
In Immigration cases:
Now-a-days many countries are imposing strict rules to those who are applying for the entry into the country. Individuals fulfill certain requirements to prove their identity by providing certain official documents such as birth certificates, marriage certificates, passports, etc. Sometimes these documents are rejected by the authorities who questions the authenticity of the documents. So in such context individual should go through DNA analysis to resolve cases such as establishing parentage of children, illegal immigration, human trafficking, etc.
In Bio-geographical ancestry analysis:
DNA fingerprinting is also helpful in identifying the ancestry of an individual. Bio-geographical ancestry analysis through DNA fingerprinting aids in solving cold cases, in cases where there is no eyewitness, in refining DNA databases, in archaeological DNA analysis, in genetic studies with forensic sensitivity, etc.
In Microbial forensics:
Microbial forensics is newly emerging area in which the analysis of human micro biome and use of microbial signatures are applied to link the crime scene exhibits with suspects. The bacteria present on human skin provides information required for forensic analyst regarding the individual host and the geographical location of host. Sequencing of micro biome in soil samples helps in differentiating varieties of species found in different locations. A multiplex real-time PCR assay using oligonucleotide mixtures targeting genomes, is specifically used for a selected group of bacteria to observe the bacterial signature for identification of biological fluids in forensic case exhibits. But a lot of research is required to use this field as an effective tool in criminal investigation.
Conclusion:
DNA fingerprinting has a tremendous impact in the criminal justice system. It plays an important role in identifying criminals. No matter what the case is, it is evident that DNA fingerprinting has revolutionized the way the world identifies biological matches. DNA fingerprinting has enormous applications in the field of forensic science that ranges from the identification of criminal in rape cases, paternity testing, and wildlife cases etc. DNA analysis is one of the evidence which is not subjected to any change in an individual’s life time, which aids in the proper identification of a person.
EDITED BY-DR. K.V.RAVI,HYDERABAD
Reference:
[1] Dr. Rukmani Krishnamurty. Forensic Biology; Selective & Scientific Books; Fourth edition 2019; PP 325-328.
[2] Wildlife Crime; Forensic working group; PP 38-46.
[3] https://bhu.ac.in/law/blj2006-072008-09/BLJ_2007/6_Pradeep_Singh.doc
[4] Brown, Terence A. Gene cloning and DNA analysis: an introduction. John Wiley & Sons, 2016. PP 311-320.
[5] Brown, Terence A. Gene cloning and DNA analysis: an introduction. John Wiley & Sons, 2016. PP 311-320.
[6] Alvarez-Cubero, M. J., et al. “Genetic identification of missing persons: DNA analysis of human remains and compromised samples.” Pathobiology 79.5 (2012): 228-238.
[7] LS, Vagish Kumar, Vina R. Vaswani, and Leena K. Pramod. “DNA analysis in identifying mass disaster victims.” IP International Journal of Forensic Medicine and Toxicological Sciences 3.3 (2018): 33-40.
[8] Budowle, Bruce, Frederick R. Bieber, and Arthur J. Eisenberg. “Forensic aspects of mass disasters: strategic considerations for DNA-based human identification.” Legal medicine 7.4 (2005): 230-243.
[9] Shrivastava, Pankaj, Toshi Jain, and V. Ben Trivedi. “DNA fingerprinting: a substantial and imperative aid to forensic investigation.” Eur J Forensic Sci 3.3 (2016): 23-30.
10.http://legaldesire.com/practical-applications-of-forensic-dna-fingerprinting
