Polymerase Chain Reaction (PCR) is a technique that functions like a molecular copying machine, allowing scientists to quickly generate millions of copies of a specific DNA segment. This amplification process is essential because it turns a minute amount of genetic material into a quantity large enough for detailed analysis. The ability of PCR to work with trace evidence has transformed forensic science, providing a powerful tool for linking individuals to crime scenes and obtaining definitive genetic profiles from samples previously considered unusable.
Why Amplification is Essential for Evidence
Crime scenes rarely contain pristine biological samples with abundant, intact DNA. Evidence often consists of degraded material or “trace DNA,” such as a few skin cells left behind after brief contact (“touch DNA”). These samples are often contaminated, exposed to environmental factors like heat and moisture, or are too small to analyze directly. Furthermore, the DNA extraction process itself can lead to a loss of genetic material, reducing the available sample size.
PCR solves this problem of quantity by targeting and exponentially multiplying specific regions of interest within the small, starting DNA template. Without this amplification, the minute amounts of DNA recovered would be undetectable by current analysis methods. By creating billions of copies from just a few starting molecules, PCR ensures sufficient material to generate a reliable genetic profile, making it possible to analyze highly compromised evidence, such as old bones or samples exposed to the elements.
The Primary Application: Analyzing Short Tandem Repeats
Forensic scientists do not analyze the entire three billion base pairs of the human genome for identification. Instead, they focus on specific, highly variable regions known as Short Tandem Repeats (STRs). An STR is a short DNA sequence, typically two to five base pairs long, that is repeated multiple times at a particular location on a chromosome. The number of times this sequence is repeated varies significantly between individuals, making STRs effective genetic markers for human identification.
PCR is used to target and amplify these specific STR loci simultaneously in a single reaction, a process called multiplexing. The primers used in the PCR are designed to bracket the STR region, meaning the resulting amplified DNA fragment (amplicon) will have a length corresponding to the number of repeats present at that locus.
Forensic analysis typically examines an established set of STR markers across the genome; in the United States, this standard set includes a minimum of 20 core autosomal loci. The power of this technique comes from analyzing multiple loci together, as the chance of two unrelated individuals having the exact same number of repeats at all 20 locations is statistically remote. By focusing on these polymorphic regions, PCR generates the unique genetic signature needed to distinguish one person from another.
Specialized Forensic PCR Techniques
While standard autosomal STR analysis is the primary method for generating a unique profile, two specialized PCR techniques are used when standard analysis is challenging or when lineage information is required.
Y-Chromosome STRs (Y-STRs)
Y-STRs are located only on the male-specific Y chromosome and track the paternal lineage, as the Y chromosome is passed directly from father to son. This analysis is particularly useful in sexual assault cases or complex mixtures where female DNA might overwhelm the small amount of male DNA from a perpetrator. Since all males in the same paternal line share the same Y-STR profile, this technique cannot uniquely identify an individual, but it can confirm paternal ancestry or exclude a suspect.
Mitochondrial DNA (mtDNA) Analysis
Mitochondrial DNA (mtDNA) analysis targets the DNA found in the cell’s mitochondria, not the nucleus. Every cell contains hundreds of mitochondria, meaning there are far more copies of mtDNA than nuclear DNA, making it useful for severely degraded samples. MtDNA is inherited exclusively from the mother, providing information about the maternal lineage. This makes mtDNA analysis the preferred method for identifying highly compromised remains, such as old bones or hair shafts lacking a root.
Interpreting the DNA Profile
After the STR loci have been amplified by PCR, the resulting DNA fragments are separated and measured using capillary electrophoresis. The fragments are moved through a fine tube under an electric field, with smaller fragments traveling faster than larger ones. A laser detects fluorescent tags attached to the amplified DNA, and the data is converted into a graph called an electropherogram.
The electropherogram displays a series of colored peaks. The position of each peak indicates the fragment’s size, which is directly related to the number of STR repeats at that locus, and the height of the peak is proportional to the quantity of DNA detected. This final set of numbers, representing the number of repeats at each tested locus, constitutes the DNA profile.
The generated profile is then compared to a known sample from a victim or suspect, or searched against national databases like the Combined DNA Index System (CODIS) in the U.S. CODIS contains profiles from convicted offenders, arrestees, and unsolved crime scenes, enabling law enforcement to link cases across jurisdictions. When a match is found, the statistical significance is calculated, providing a measure of the probability that a random, unrelated person would have the identical profile.