The invention of the Polymerase Chain Reaction (PCR) revolutionized the field of genetic analysis. PCR is a molecular technique that allows scientists to create millions or even billions of copies of a specific DNA segment from a minute starting sample. This copying capability was the necessary catalyst for modern DNA profiling. DNA profiling is a powerful method used for individual identification by examining unique patterns within a person’s genetic code. The development of PCR transformed DNA profiling from a slow, resource-intensive procedure into a rapid, highly sensitive, and routine forensic tool.
The Core Principle of DNA Fingerprinting
The basis of individual DNA identification lies in the small amount of genetic material that varies between people. While over 99.9% of the human genome is identical among unrelated individuals, scientists focus on specific, highly variable regions to create a unique profile. These target locations are known as polymorphic markers.
The current standard for DNA profiling concentrates on Short Tandem Repeats (STRs), which are sequences of 2 to 6 base pairs repeated multiple times at specific points on a chromosome. For example, a sequence like “GATA” might repeat 7 times in one person and 11 times in another at the same location. Since every person inherits one copy of DNA from each parent, they possess two alleles, or repeat counts, for each STR location.
The individuality of the profile is established by examining numerous STR loci across the genome, typically 20 or more in forensic analysis. The statistical probability of two unrelated individuals sharing the exact same number of repeats at all tested locations is vanishingly small.
The Pre-PCR Problem: Insufficient DNA Sample
Before the advent of PCR, the primary method for analyzing DNA variation was Restriction Fragment Length Polymorphism (RFLP) analysis. This technique required the initial DNA sample to be large and relatively intact to yield a measurable result. RFLP analysis typically needed micrograms of high-quality DNA.
Forensic samples collected at crime scenes, such as a few cells from a touch or a faint blood smear, often contain only picograms of DNA, which is a thousand times less than the required amount. Furthermore, environmental factors like moisture, heat, or bacteria can quickly degrade the DNA into tiny, fragmented pieces. Pre-PCR methods were simply too insensitive to work with these trace or compromised samples.
The limitations of RFLP severely restricted the practical application of DNA fingerprinting in forensic science and other fields like archaeology. Most real-world evidence simply did not provide the necessary quantity or quality of genetic material to generate a profile. This inability to analyze small or degraded samples created a significant technological barrier.
Polymerase Chain Reaction: A Targeted Copying Machine
The Polymerase Chain Reaction solved the fundamental problem of sample quantity. This technique uses a cyclical process of heating and cooling to rapidly synthesize millions of copies of a specific DNA segment. The initial step, denaturation, involves heating the double-stranded DNA to approximately 95°C to break the bonds holding the two strands together, separating them.
The temperature is then lowered to the annealing phase, typically between 50°C and 65°C. This cooling allows short, lab-designed DNA sequences, called primers, to attach specifically to the beginning and end points of the target STR regions. These primers ensure that only the relevant, highly variable sections of the genome are copied, ignoring non-target DNA.
Following primer binding, the temperature is raised to about 72°C for the extension step, where a heat-stable enzyme called Taq polymerase begins adding nucleotides. The enzyme uses the separated single strand as a template to build a new complementary DNA strand, starting from the bound primer. This three-step cycle is repeated 28 to 32 times, resulting in an exponential amplification of the target DNA.
Each cycle doubles the number of target DNA molecules, converting an invisible trace sample into a quantity sufficient for analysis within a few hours. The specificity of the primers means that even if the original DNA is degraded, the PCR process can still successfully amplify the small, intact STR regions. This capability made the analysis of compromised forensic evidence possible for the first time.
Enabling Analysis: How PCR Makes Fingerprinting Practical
The massive quantity of target DNA produced by PCR is the prerequisite for the final analysis, which determines the individual’s genetic profile. Modern DNA profiling typically uses a technique called multiplex PCR, which simultaneously amplifies all the required STR loci in a single reaction tube. The primers used in this process are manufactured with fluorescent tags attached to them.
After amplification, the DNA fragments are separated and measured using a method called capillary electrophoresis. The amplified STR fragments are pulled through a fine glass tube containing a gel-like polymer. Smaller fragments travel faster through this matrix than larger ones.
As the fragments pass a detection window, a laser excites the fluorescent tags, and a detector records the light signal. This process generates an electropherogram, which is a graphical output where distinct peaks correspond to the different lengths of the STR fragments. By comparing the size of each peak against a known reference standard, scientists can precisely determine the number of repeats at every analyzed locus. This precise measurement of the length variations translates the genetic material into the unique sequence of numbers that forms the final, reliable DNA fingerprint.