Difference Between RAPD and RFLP

Key Difference – RAPD vs RFLP
 

Genetic markers are used in Molecular Biology to identify genetic variations between individuals and species. Random Amplified Polymorphic DNA (RAPD) and Restriction Fragment Length Polymorphism (RFLP) are two important molecular markers routinely used in laboratories. RAPD is performed with short and arbitrary oligonucleotide primers, and it is based on the random amplification of the multiple locations throughout the template DNA of the organism.  RFLP is performed with a specific restriction endonuclease, and it is based on the polymorphism of resulted restriction fragments and hybridization. The key difference between RAPL and RFLP is that RAPD is a type of PCR technique performed without the prior sequence knowledge whereas RFLP is not involved in PCR and requires prior sequence knowledge to carry out the technique.

CONTENTS
1. Overview and Key Difference
2. What is RAPD
3. What is RFLP
4. Side by Side Comparison – RAPD vs RFLP
5. Summary

What is RAPD?

RAPD is a useful molecular marker in molecular biology. It is a quick and easy technique. RAPD can be defined as a method which results in polymorphic DNA sequences as a result of random amplification of multiple locations of the target DNA template. RAPD uses short oligonucleotide primers with arbitrary sequences for the PCR amplification. Primers are artificially synthesized without the prior sequence knowledge. Hence, it is considered as an easy and useful technique.

The following major steps are involved in RAPD.

1. Extraction of target DNA
2. Amplification of the multiple locations of the target DNA using randomly chosen primers
3. Gel electrophoresis of the amplified PCR products
4. Staining with ethidium bromide and identification of the polymorphism

As a result of the variation in the primer annealing, different fragments with different lengths are generated during the amplification. Hence, banding patterns on the gels are different among individuals and species. Thus, RAPD enables the detection of genetic variation among organisms in identification and differentiation.

RAPD is applied in various studies of molecular biology such as identification of the genetic difference between closely related species, gene mapping, DNA fingerprinting, identification of inherited diseases, etc.

What is RFLP?

Restriction Fragment Length Polymorphisms (RFLPs) is a molecular marker used in molecular biology for the identification of genetic variation in homologous DNA sequences. It is the first genetic marker developed for DNA fingerprinting. All organisms produce unique DNA profiles when restricted with specific restriction enzymes. RFLP serves as an important tool in producing unique DNA profiles of individuals and detection of genetic variation among them. When DNA samples are digested with specific restriction endonucleases, it yields different DNA profiles which are unique to each individual. Therefore, the principal of this method is the detection of genetic variation among organisms by restricting homologous DNA with specific restriction enzymes and analysis of the fragment length polymorphism via gel electrophoresis and blotting. Blotting patterns are unique to each organism and characterize the specific genotypes.

Following steps are involved with RFLP.

1. Isolation of sufficient amount of DNA from samples
2. Fragmentation of the DNA samples with specific restriction endonucleases into short sequence
3. Separation of the resulted fragments with different lengths by agarose gel electrophoresis.
4. Transfer of the gel profile into a membrane by Southern blotting
5. Hybridization of the membrane with labeled probes and analysis of the fragment length polymorphism in each profile

RFLP has various applications such as diagnosis of inheritance diseases, genome mapping, criminal identification in forensic studies, paternity testing, etc.

What is the difference between RAPD and RFLP?

RAPD vs RFLP
RAPD is a molecular marker based on random primers and PCR.	RFLP is a molecular marker based on the production of different length restriction fragments.
Required Sample
Small DNA samples are enough for the RAPD analysis.	A large amount of extracted DNA sample is required for RFLP analysis.
Time
RAPD is a quick process.	RFLP is a time-consuming process.
Primer Use
Random primers are used and same primers can be used for different species.	Species-specific probes are used in RFLP for hybridization.
Reliability
Reliability of the technique is less compared to RFLP.	RFLP is a reliable technique.
Blotting
RAPD involves southern blotting.	Southern blotting is a one step of RFLP.
Detection of Allelic Variation
Allelic variations cannot be detected by RAPD.	Allelic variations can be detected by RFLP.
Need for Sequence Knowledge
RAPD does not require prior sequence knowledge.	Prior sequence knowledge is required for probe designing.
PCR
PCR is involved with RAPD	PCR is not involved with RFLP.
Reproducibility
RAPD has a low reproducibility	RFLP is has a high reproducibility compared to RAPD.

Summary – RAPD vs RFLP

RAPD and RFLP are important markers used in molecular biology. Both methods are capable of detecting genetic variation among organisms. RAPD is performed using random primers. RFLP is performed using specific restriction enzymes. Both methods produce DNA profiles unique to individual organisms. RAPD is involved comparably few steps than RFLP. But it produces less reliable and reproducible results than RFLP. This is the main difference between RAPD and RFLP.

References:
1. Dos, J. B., J. Nienhuis, P. Skroch, J. Tivang, and M. K. Slocum. “Comparison of RAPD and RFLP genetic markers in determining genetic similarity among Brassica oleracea L. genotypes.” TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik. U.S. National Library of Medicine, Mar. 1994. Web. 12 Apr. 2017
2. Powell, Wayne, Michele Morgante, Chaz Andre, Michael Hanafey, Julie Vogel, Scott Tingey, and Antoni Rafalski. “The comparison of RFLP, RAPD, AFLP

Image Courtesy:
1. “RFLP genotyping” By (unknown) – National Institutes of Health (Public Domain) via Commons Wikimedia