Restriction Fragment Length Polymorphism (RFLP) is a foundational technique in molecular biology used to identify variations in the DNA sequence of different individuals. The method detects these differences by analyzing the distinct lengths of DNA fragments produced after treating the DNA with specific enzymes. RFLP was historically the first technology capable of generating unique DNA profiles for widespread application, allowing researchers to study genetic differences and track the inheritance of specific DNA regions.
The Role of Restriction Enzymes
The “R” in RFLP stands for Restriction, referring to specialized enzymes that serve as molecular scissors. Restriction enzymes, or restriction endonucleases, are naturally occurring proteins isolated primarily from bacteria. Their biological purpose is to protect the host cell by cutting up foreign DNA. These enzymes recognize and bind to very specific, short sequences of nucleotides, known as restriction sites, along the DNA molecule.
A typical restriction site is a palindromic sequence, reading the same forward on one DNA strand as it does backward on the complementary strand. Once the enzyme recognizes its specific site, it cleaves the phosphodiester bonds of the DNA backbone. This precise cutting action fragments the long strands of DNA into smaller pieces. The choice of restriction enzyme determines the exact points at which the DNA will be fragmented for analysis.
Performing the RFLP Test
The RFLP test is a multi-step laboratory process that begins with the purification of an intact DNA sample. This isolated DNA is subjected to a restriction digest, where it is incubated with the chosen restriction enzyme, resulting in millions of DNA fragments. The next step is gel electrophoresis, a technique that separates these DNA pieces based on their size and electrical charge.
During gel electrophoresis, the fragmented DNA is loaded into an agarose gel matrix, and an electric current is applied. Since all DNA fragments carry a negative charge, they migrate toward the positive electrode. Shorter fragments travel farther than the longer fragments, effectively sorting the entire collection by length. This separation, however, produces a smear of fragments that is impossible to interpret directly.
To visualize only the relevant fragments, Southern Blotting is performed. This involves transferring the size-separated DNA fragments onto a solid nylon membrane, fixing them in their separated positions. The membrane is subsequently incubated with a labeled probe—a short, single-stranded piece of DNA designed to bind only to the specific DNA sequence of interest. This hybridization highlights the fragments complementary to the probe. Finally, the membrane is exposed to an imaging system to produce an autoradiogram, which reveals a distinct pattern of bands representing the lengths of the targeted DNA fragments.
Genetic Basis for Fragment Length Variation
The “LP” in RFLP stands for Length Polymorphism, describing the underlying genetic differences that create the unique banding patterns. When two individuals’ DNA samples are treated with the same restriction enzyme, they may yield different fragment lengths if there is a variation in the location of a restriction site.
The simplest mechanism for this variation is a Single Nucleotide Polymorphism (SNP) that either creates a new restriction enzyme recognition site or destroys an existing one. For example, if a base pair change eliminates a restriction site, the enzyme will no longer cut at that position, resulting in one long fragment instead of two shorter ones. Conversely, a mutation that generates a new site results in two smaller fragments where there was previously one large one.
Other genetic changes, such as insertions, deletions, or variations in the number of short, repeated DNA sequences (like Variable Number Tandem Repeats or VNTRs), can also alter the distance between two restriction sites. This change in distance directly translates to a difference in the length of the resulting DNA fragment. Because these variations are inherited, the resulting RFLP pattern is unique to an individual, providing a genetic fingerprint that can be tracked through families or compared between unrelated individuals.
Key Uses of RFLP Technology
RFLP technology provided the first reliable method for genetic individualization, revolutionizing several fields. Its application in forensic science established the concept of DNA fingerprinting by allowing the comparison of DNA from a crime scene sample to that of a suspect. The unique banding pattern serves as a highly discriminatory genetic signature, instrumental in early criminal investigations.
The inherited nature of RFLP markers made the technique an excellent tool for paternity testing. A child’s pattern is compared to the patterns of the alleged father and the mother. By showing that approximately half of the child’s bands were inherited from the father, RFLP provided strong biological evidence of a parental relationship.
The technology was also instrumental in early efforts to map the human genome by serving as genetic markers. Researchers used RFLP markers to track the inheritance of specific DNA segments linked to inherited disorders within families, helping to localize the genes responsible for diseases like Huntington’s and Cystic Fibrosis.
Although RFLP has largely been replaced in modern forensic and clinical labs by faster, more sensitive polymerase chain reaction (PCR)-based methods, its historical importance as the original technique for large-scale genetic analysis remains undisputed.