How RFLP works ?

Restriction Fragment Length Polymorphism (RFLP) is a molecular biology technique used to detect variations in DNA sequences among individuals. RFLP analysis relies on differences in the lengths of DNA fragments generated by restriction enzymes cutting at specific recognition sites within the DNA sequence.

 

Steps

DNA Extraction: DNA is extracted from the sample of interest, such as plant tissue or blood.

 

Digestion with Restriction Enzymes: The extracted DNA is then digested with one or more restriction enzymes, which recognize specific DNA sequences (restriction sites) and cleave the DNA at those sites. Different restriction enzymes recognize different DNA sequences, leading to the generation of DNA fragments of varying lengths.

 

Electrophoresis: The digested DNA fragments are separated by size using gel electrophoresis. The DNA fragments are loaded into wells in an agarose gel and subjected to an electric field. Smaller fragments move more quickly through the gel, while larger fragments move more slowly.

 

Southern Blotting (Optional): In some cases, the separated DNA fragments may be transferred from the gel onto a membrane in a process called Southern blotting. This step is often used for further analysis or to transfer the DNA fragments to a membrane for hybridization with labeled probes.

 

Hybridization: The separated DNA fragments may be hybridized with labeled probes that are complementary to specific DNA sequences of interest. The probes bind to complementary sequences within the DNA fragments, allowing for the detection of specific DNA sequences.

 

Detection: The DNA fragments are visualized using various detection methods, depending on whether radioactive or non-radioactive probes are used. Autoradiography or chemiluminescence is often employed for radioactive probes, while fluorescence detection is used for non-radioactive probes.

 

Analysis: The pattern of DNA fragments generated by RFLP analysis is analyzed to detect polymorphisms (variations) in the DNA sequence. Polymorphisms may be due to differences in the presence or absence of restriction sites, leading to differences in fragment lengths between individuals.

 

RFLP analysis was one of the earliest molecular techniques used for genetic analysis and has been widely used in genetic mapping, paternity testing, forensic analysis, and genetic diversity studies. While newer techniques such as polymerase chain reaction (PCR) and DNA sequencing have largely replaced RFLP analysis in many applications, it remains an important tool in certain areas of genetic research.