Human identification requires distinguishing one person’s genetic blueprint from another’s, even though the vast majority of human DNA is identical across all individuals. This process relies on identifying minute differences in the genetic code using two molecular biology techniques: Polymerase Chain Reaction (PCR) and Gel Electrophoresis. This sequential system allows scientists to analyze extremely small or degraded genetic samples, such as those found at a crime scene. The overall goal is to create a unique genetic profile that serves as an individual biological identifier.
Targeting the Code: Short Tandem Repeats
Scientists focus on specific, highly variable areas of the genome to create a distinctive profile, rather than analyzing the entire three billion base pairs of human DNA. These target regions are called Short Tandem Repeats (STRs), which are repeating, non-coding DNA segments scattered throughout the chromosomes. The repeat units within STRs are very short, typically ranging from two to seven base pairs in length.
The defining feature of STRs is that the number of times the core sequence is repeated varies significantly between individuals. For example, one person might have 10 repeats of a sequence at a specific location, while another might have 15 repeats at the exact same location. This difference in the number of repeats means that the length of the STR region is unique to each person.
STRs are located in non-coding regions of the genome, meaning they do not determine traits or characteristics, making them ideal for identification purposes. Analyzing multiple STR locations, or loci, provides a strong statistical method for discrimination. The probability of two unrelated people having the exact same number of repeats at every analyzed location is extremely low. Current forensic standards examine over 20 different STR loci to ensure the resulting DNA profile is essentially unique.
Making Copies: The Function of PCR
The amount of DNA recovered from a biological sample is often insufficient for direct analysis, making the amplification of the target STR regions necessary. This is the function of the Polymerase Chain Reaction (PCR), a technique that creates millions to billions of copies of a specific DNA segment quickly. PCR works by cycling a reaction mixture through three distinct temperature-dependent steps inside an instrument called a thermal cycler.
The process begins with denaturation, where the double-stranded DNA template is heated to separate the two strands. Next, in the annealing step, the temperature is lowered, allowing short, synthetic DNA molecules called primers to bind to the ends of the single-stranded target STR regions. These primers are specifically designed to flank the STR sequence of interest, ensuring only the variable region is copied.
The final step is extension, where the temperature is raised again for a heat-stable enzyme, DNA polymerase, to begin synthesizing new DNA strands. Starting from the attached primers, the enzyme adds complementary nucleotides to the template strand. Repeating this three-step cycle approximately 25 to 35 times leads to an exponential increase in the amount of STR DNA. This transforms an invisible trace amount into a quantity large enough to be detected and analyzed.
Sorting by Size: Visualizing DNA with Gel Electrophoresis
Once the STR regions have been amplified by PCR, the next step is to separate these fragments based on their length using Gel Electrophoresis. This technique uses an electric field to push the negatively charged DNA molecules through a porous, gel-like matrix. The gel acts like a molecular sieve, impeding the movement of the DNA fragments as they migrate.
The amplified DNA samples are loaded into small wells at one end of the gel, which is connected to a power supply. Since DNA molecules carry a uniform negative charge, they are pulled toward the opposite end of the gel by the electric field. Shorter DNA fragments pass more easily through the gel’s pores and therefore travel a greater distance in a given amount of time.
Conversely, longer DNA fragments, which correspond to STR regions with more repeats, are slowed down by the gel matrix and migrate a shorter distance. This size-based separation results in a distinct pattern of bands or peaks, with each band representing an STR fragment of a specific length. Scientists determine the size of the amplified STRs by comparing the migration distance of the sample fragments to a set of DNA fragments of known lengths.
The Final Match: Creating a Unique DNA Profile
The final stage involves interpreting the size-separated STR fragments to construct a unique genetic identifier known as a DNA profile. The specific number of base pairs for each STR fragment is recorded, representing the number of repeats at that particular locus. Since humans inherit one copy of each chromosome from each parent, an individual will have two values—one from each parent—for every STR locus analyzed.
The resulting DNA profile is a numerical code that lists the length of the STR fragments across all the analyzed loci. This profile is used for practical applications, such as forensic testing, where a crime scene sample is compared against a suspect’s profile or a database. A match is declared when the numerical values for the STR repeats align perfectly across all tested loci between two samples, confirming they originated from the same individual.
This combined technique is also routinely used to confirm biological relationships, such as in paternity testing, by comparing inherited STR values. The profile is also used in identifying human remains or disaster victims when visual identification is impossible. By analyzing multiple, independent STR markers, the likelihood of two unrelated people sharing the exact same profile becomes statistically minute.