RFLP based DNA profiling
Restriction Fragment Length Polymorphism (RFLP) was first discovered in 1984 by a British geneticist and a professor at the University of Leicester, Sir Alec Jeffreys. The concept was based on the observation that different individuals have unique patterns of fragments with varying lengths when their DNA is cut with specific restriction enzymes. This variation in the length of DNA fragments provides a way to distinguish between individuals, also known as ‘DNA profiling’ or DNA fingerprinting’. RFLP-based DNA profiling was a breakthrough in forensic science and helped revolutionize criminal investigations.
What are Restriction Enzymes?
Restriction enzymes (also called restriction endonucleases) are proteins that are found in bacteria that have the ability to cleave DNA at specific sequences, called restriction sites or recognition sites. These restriction sites have palindromic sequences (typically 4 to 8 base pairs in length), which means that the sequence of base pairs in a certain direction (e.g. 5' to 3') on one strand is identical to the sequence in the same direction (e.g. 5' to 3') on the complementary strand.
Restriction enzymes play an important role in the defense mechanism of bacteria against foreign DNA, such as viral DNA. They recognize these sites and cut the foreign DNA, thus, preventing it from replicating and infecting the bacterium. In molecular biology, restriction enzymes isolated from bacteria are widely used as tools to cut DNA into fragments for a variety of applications, including RFLP analysis.
The restriction enzymes can be categorized based on the length and sequence of their recognition sites. There are many different types of restriction enzymes, each with a unique recognition site and cutting pattern, allowing for the production of a wide variety of DNA fragment patterns.
For example: EcoRI is a type of restriction enzyme that recognizes the sequence 5'-GAATTC-3' and cleaves the DNA molecule at that site by creating a break in the sugar-phosphate backbone of the DNA molecule. This cleavage results in the production of two separate DNA fragments, each with a single-stranded overhang or ‘sticky end’.
Figure: Restriction of DNA fragment by EcoRI
What are Sticky ends?
The ‘sticky ends’ produced by restriction enzymes are single-stranded overhangs on the ends of DNA fragments that can participate in hydrogen bonding with complementary overhangs on other DNA molecules. This property of sticky ends makes them useful tools in molecular biology for the manipulation and analysis of DNA.
One of the main uses of sticky ends is in DNA cloning, where they allow for the specific and efficient joining of two DNA fragments. For example: A restriction enzyme can be used to cleave the target DNA and the vector DNA at specific recognition sites, creating complementary sticky ends. The target DNA and vector DNA can then be mixed together and joined using an enzyme ‘ligase’, creating recombinant DNA.
Procedure for RFLP-based DNA profiling
The steps involved in performing RFLP analysis are:
DNA extraction: The first step is to extract the DNA from the biological sample. This can be done using a variety of methods, such as phenol-chloroform extraction, salting out, or by using commercially available DNA extraction kits.
Restriction Enzyme Digestion: The extracted DNA is then cut into fragments using restriction enzymes. During this process, a restriction enzyme is added to a mixture containing the target DNA and a buffer solution to maintain optimal conditions for the reaction. The restriction enzyme will then bind to the specific restriction sites and cleave the phosphodiester bonds, generating fragments of DNA. The length and number of fragments generated will depend on the specific restriction enzyme used and the number of restriction sites present in the DNA, thus offering a method for distinguishing between individuals.
Gel Electrophoresis: The DNA fragments are then separated based on size using gel electrophoresis. The technique involves placing the DNA sample in a gel matrix (made up of a porous polymer such as agarose or polyacrylamide) and subjected to an electric field. This allows the negatively charged DNA to move towards the positively charged electrode. Smaller DNA fragments will move faster through the gel than larger fragments, resulting in the separation of the DNA sample into distinct bands based on size.
Transfer: The separated DNA fragments are then transferred to a piece of nitrocellulose or nylon membrane using a technique called Southern blotting. This technique involves first placing the gel in contact with the nitrocellulose or nylon membrane, and then applying pressure to force the DNA fragments out of the gel and onto the membrane. This step is necessary to immobilize and stabilize the DNA fragments and make them accessible for probing with a labelled DNA sequence.
Hybridization with a Probe: The transferred DNA fragments are then incubated with a labelled probe, which binds or ‘hybridizes’ to a specific sequence of DNA. A probe is a fragment of single-stranded DNA (usually 100-1000 bp long) that can be labelled with a radioactive or non-radioactive marker (such as a fluorescent dye or a chemiluminescent molecule), to detect the presence of DNA sequences that are complementary to the sequence in the probe. After hybridization, the membrane is washed to remove any unbound and un-specifically bound probes.
Visualization: After hybridization with a probe, DNA fragments on a membrane can be detected using various visualization methods based on the probe label. A radioactive label can be detected using X-ray film to produce an autoradiogram that shows the location of the labelled DNA fragments. Similarly, a fluorescent or chemiluminescent label can be detected using a gel imaging system or gel documentation system, which produces an image of the labelled DNA fragments. The pattern of bands can be analyzed to identify the DNA fragments present in the sample.
Data Analysis: The final step is to analyze the data and interpret the results by comparing the banding patterns between different samples, which can provide insights into the DNA samples being tested. For instance, if two samples have very similar banding patterns, it indicates that they share a high degree of genetic similarity. Conversely, if two samples have distinct banding patterns, it suggests a low degree of genetic similarity. Additionally, if two samples have an identical banding pattern, it may indicate that they are from the same individual or identical twins.
The interpretation of the results obtained from RFLP analysis can be utilized for various applications. In forensic investigations, RFLP analysis can be used to compare DNA samples from crime scenes to those from suspects or victims, which can provide valuable evidence to aid in criminal investigations.
Limitations of RFLP analysis
RFLP was a popular technique for DNA profiling in the early days of forensic science. However, it had several limitations that made it challenging to use in practice. Firstly, it required a relatively large amount of high-quality DNA, which could be difficult to obtain from degraded or mixed samples. Secondly, RFLP analysis was time-consuming and labor-intensive, involving multiple steps of gel electrophoresis and Southern blotting. Moreover, the interpretation of RFLP patterns could be subjective, leading to potential errors and controversies in forensic investigations.
Over the period of years, Short Tandem Repeats (STRs) have emerged as a more practical and reliable alternative for DNA profiling.