In the pursuit of conducting a thorough investigation into a crime scene and apprehending the perpetrator, DNA analysis stands as a highly valuable and reliable tool. DNA technology came into existence in the early 1980s. Before that, individualization and identification were done based on blood group types known to scientists. In 1984, Alec Jeffreys developed the method of DNA profiling, also known as DNA fingerprinting, which revolutionized crime investigation. Since then, there has been no looking back. The only case where DNA profiling fails to answer an investigator’s query is in the case of monozygotic (identical) twins since they share the same DNA. In such cases, fingerprints are the only discriminatory aspect to identify the real offender.
Understanding DNA Fingerprinting
DNA fingerprinting targets determining the specific and unique sequence of nucleotides in a particular and very specific part of the DNA, which is unique to every individual. It is commonly believed that 99% of the genetic DNA composition is the same in all individuals, and it is only the 1% variation that has given rise to billions of unique individuals all around the globe.
Basic Concepts Related to DNA Fingerprinting
DNA consists of a coding region (that codes for specific proteins contributing to the external and internal characteristics of an individual) and a non-coding region (that does not code for any protein and makes up 95% of the genetic DNA, also called junk DNA). In these non-coding regions exist certain unique inheritable sequences of 1-6 base pairs or 9-80 base pairs that repeat in tandem. These repetitive sequences are known as Short Tandem Repeats (STRs) and Variable Number Tandem Repeats (VNTRs). These tandem repeats, also called Genetic Markers, can be separated from the remaining mass of DNA by density gradient centrifugation.

These STRs and VNTRs are polymorphic, meaning every individual has unique tandem repeats in their genetic DNA. This polymorphism is due to variations from genetic mutations. Therefore, every individual has a unique number of tandem repeats at a particular locus on the chromosome.
Each offspring inherits 50% of their genetic DNA from their mother and 50% from their father. However, the tandem repeats that the offspring possess will be unique and different from those of their parents due to deletion, mutation, or insertion. This variation forms the basic principle of DNA fingerprinting.
Recognition sites are palindromic sequences of 2-5 base pair lengths present on the DNA. These sites exist for recognition by restriction enzymes, which cleave the DNA into small, irregular length fragments.
An Overview of Sanger Sequencing
Sanger Sequencing, also known as the chain termination method, was developed by Fredrick Sanger in 1977 to determine nucleotide sequences in genetic DNA. In this technique, the double helical DNA is denatured into single-stranded DNA, and the specific target DNA sequence is used as a template. In addition to normal dNTPs, a very low concentration of dideoxyribonucleotides (ddNTPs – ddATP, ddCTP, ddTTP, ddGTP) is added. The OH group at the 3’ end of these ddNTPs is absent, so during the polymerization of the template strand into a double helix, when these ddNTPs are added to the target sequence at random, the polymerization reaction terminates at any random length.
In manual Sanger Sequencing, four different tubes are used, each for ddATP, ddGTP, ddCTP, and ddTTP. In automated Sanger Sequencing, a single tube is used with all the ddNTPs mixed together, and all the dNTPs have a unique colored fluorescent label.
The different lengths of terminated chains are then run on gel electrophoresis to separate the DNA fragments based on their lengths, and a radiogram is created for reading and analyzing the Sanger sequence. In automated sequencing, the fragments containing the radiolabeled dNTPs are subjected to capillary electrophoresis, where the capillary is equipped with a charged couple device (CCD) that detects fluorescence signals to create the Sanger sequence graph.
The Sanger sequence is read manually by reading all four wells simultaneously, starting from the bottom and going upwards. In automated sequencing, the computer reads individual bands of the capillary gel to identify the terminal ddNTP.
An Overview of Next Generation Sequencing
The Human Genome Project, which ultimately decoded all human genes, used the Sanger Sequencing method to decode all information in the human genome. It began in 1990 and completed in 2022. With technological advancements, this feat can now be achieved in only one day. Next Generation Sequencing (NGS), also known as massively parallel sequencing, can sequence billions of DNA strands at once and can also sequence RNA.
NGS uses adapter molecules binding to both ends of DNA fragments. The most popular instrument for NGS sequencing is Illumina, which uses a method called sequencing by synthesis. The main advantage of NGS is that it can determine the sequence and order of nucleotides of either the entire genome or any targeted region of the DNA or RNA.
The Impact of NGS Technology in Forensic Science
The discriminatory power of NGS technology can be beneficially exploited in forensic science. The main goal of forensic DNA examination is either to narrow down suspects in a crime or to specifically prove the guilt of the perpetrator in court by using the highly polymorphic nature of STRs and VNTRs in the non-coding region of the DNA. Existing DNA technology works on identifying 26+1 highly polymorphic genetic markers on the genomic DNA and the STRs of the X and Y chromosomes. With the development of NGS technology, determining the nucleotide sequence of the entire genome or specific markers present in the DNA extracted from biological evidence found at any crime scene would help investigators accurately identify the perpetrator and secure their conviction in court.
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