In this blog post, we will summarize the principles of DNA fingerprinting, how its main methods—RFLP and PCR—work, their advantages and disadvantages, and real-world applications.
September 11, 2001, was a nightmarish day for Americans. Members of Al-Qaeda, a fundamentalist Muslim group led by Osama bin Laden, hijacked four airplanes that morning and crashed two of them into the Twin Towers of the World Trade Center in New York; within eight hours, the Twin Towers had collapsed in a devastating manner. Countless people inside the buildings died instantly, and many more, including rescue workers, lost their lives as the structures collapsed. This terrorist attack claimed over 3,000 lives; while most of the victims were so badly damaged that their bodies were unrecognizable, the scientific technique of DNA fingerprinting made it possible to identify approximately 1,700 of them and return their remains to their families.
Every human cell contains chromosomes, which are long strands of DNA tightly coiled around a double helix composed of four nucleotides: A (adenine), G (guanine), C (cytosine), and T (thymine). DNA encodes the amino acid information necessary for producing various proteins using the four nucleotides A, C, G, and T, serving as a reference for cells when synthesizing proteins. While approximately 99% of human DNA is identical, there is about a 1% difference between individuals. Since the entire DNA consists of about 3 billion base pairs, even a 1% difference can result in numerous variations in the sequence. These differences can be used like an identification tag to distinguish individuals, and since DNA is present in every cell, it can be obtained even from minute samples such as a strand of hair, a small bone fragment, or saliva. Thanks to these characteristics and advances in life science technology, DNA is widely used for identifying suspects, confirming paternity, and identifying unidentified remains; this is known as “DNA fingerprinting.”
DNA fingerprinting used in forensic science can be broadly categorized into methods utilizing Restriction Fragment Length Polymorphism (RFLP) and the Polymerase Chain Reaction (PCR).
RFLP analysis uses proteins called restriction enzymes. Restriction enzymes cut the DNA double strand at specific nucleotide sequences. For example, one restriction enzyme cuts at the “ACCTGG” sequence, while another cuts at a different sequence such as “CGTCCGC.” Human DNA contains regions that do not encode proteins, known as junk DNA. Genes that encode important proteins are eliminated because mutations in them are detrimental to survival, but mutations in junk DNA do not adversely affect offspring and are therefore passed down unchanged to future generations. For this reason, mutations accumulate in junk DNA, resulting in different nucleotide sequence patterns among individuals.
By utilizing these individual sequence differences, when DNA is treated with a restriction enzyme that recognizes and cuts only specific sequences, the lengths of the resulting fragments vary from person to person. For example, one person might have three specific cleavage sites, while another might have ten; when treated with the same restriction enzyme, the distribution of fragment lengths differs. When the resulting fragments are separated by electrophoresis, they migrate different distances depending on their length, and individuals can be identified by comparing the resulting band patterns.
RFLP has the advantage of allowing rapid identification of individuals without analyzing the entire 3 billion-base sequence, but it has the disadvantage of being difficult to apply to samples with low DNA quantities or those that have been chemically degraded. RFLP may be unsuitable when only a small amount of DNA is available from a crime scene.
PCR is the method that overcomes these limitations. PCR is a technique that uses a polymerase enzyme to selectively amplify specific DNA segments in large quantities. A single strand of DNA consists of four nucleotides—A, C, G, and T—linked together like a chain, with A pairing with T and C pairing with G. The double-stranded DNA is separated by heat to form two strands, and primers—short DNA fragments corresponding to the starting points on both sides of the region to be replicated—are added. When the temperature is lowered, the polymerase binds to the primers and synthesizes the DNA in that region. By repeating the process of heating to separate the newly formed strands and then lowering the temperature, the DNA in the target region is amplified exponentially.
PCR is highly useful for analyzing small amounts of DNA because it can amplify even minute quantities of genetic material into large amounts and can be used even with partially degraded samples. However, it has the disadvantage of being susceptible to sample contamination. If other DNA is mixed into the sample, it will be amplified along with the target DNA, potentially distorting the analysis results.
DNA fingerprinting has been instrumental in solving many problems that were previously unsolvable. Its use in criminal investigations dates back to the 1980s, when Colin Pitchfork was the first person to be convicted based on DNA fingerprinting. In South Korea, DNA fingerprinting was also used on cloned animals to determine whether Professor Hwang Woo-suk’s research team had fabricated data.
However, DNA fingerprinting also has its vulnerabilities. In the 1995 O. J. Simpson murder trial, the prosecution submitted Simpson’s DNA found at the crime scene as evidence; however, the defense pointed out issues in the DNA collection, labeling, and testing processes, as well as the possibility of contamination. These concerns were acknowledged, and the evidence was not admitted as conclusive proof. As such, contamination that can occur during sample handling is a critical aspect that must be carefully managed in DNA fingerprinting.
