Random amplified polymorphic DNA (RAPD) is a type of PCR that uses short, arbitrary primers to randomly amplify DNA fragments. Several primers are used in PCR with genomic DNA to generate unique band patterns. RAPD does not require prior knowledge of DNA sequences and can detect mutations if they occur where the primer binds. However, it has lower resolution than targeted methods and results can be difficult to interpret. RAPD analyzes 100 to 3000 base pair fragments and computer programs can analyze the profiles.

1. Random amplified Polymorphic DNA
(RAPD)
DR RADHAKRISHNA G PILLAI
Department of Life Sciences
University of Calicut
Kerala, INDIA 673 635

2. RAPD
• RAPD (pronounced "rapid") stands for 'Random
Amplified Polymorphic DNA‘
• It is a type of PCR reaction, but the segments of DNA
that are amplified are random
• Several arbitrary, short primers (8–12 nucleotides)
are used
• PCR using a large template of genomic DNA
• Fragments may amplify
• A Pattern OF PCR product obtained
• A semi-unique profile can be obtained from a RAPD
process

3. Uniqueness of RAPD
• No knowledge of the DNA sequence for the
targeted genome is needed as the primers bind
random
• This makes the method popular for comparing
the DNA of biological systems that are not well
studied
• Because it relies on a large, intact DNA template
sequence

4. RAPD
• No fragment is produced if primers annealed too far
apart or 3' ends of the primers are not facing each
other
• If a mutation has occurred in the template DNA at the
site that was previously complementary to the primer,
a PCR product will not be produced, resulting in a
different pattern of amplified DNA segments on the
gel
• Selecting the right sequence for the primer is very
important
• Different sequences will produce different band
patterns
• More specific recognition of individual strains by
different primers

5. Limitations of RAPD
• Resolving power is much lower than targeted, species
specific DNA comparison methods, such as short tandem
repeats.
• Nearly all RAPD markers are dominant, i.e. it is not possible
to distinguish whether a DNA segment is amplified from a
locus that is heterozygous (1 copy) or homozygous (2 copies)
• Codominant RAPD markers, observed as different-sized DNA
segments amplified from the same locus, are detected only
rarely.
• RAPD technique is laboratory dependent and needs
carefully developed laboratory protocols to be reproducible.
• Mismatches between the primer and the template may
result in the total absence of PCR product as well as in a
merely decreased amount of the product. Thus, the RAPD
results can be difficult to interpret

7. RAPD
• 100 to 3000 bp depending upon the genomic
DNA and the primer
• This technique is sensitive, fast, requires the
use of no radioactive probes, and is easily
performed.
• Computer programs available to analyse
RAPD profile

8. Amplified Fragment Length Polymorphism
(AFLP)
• AFLP is A PCR based tool used in genetics research, DNA
fingerprinting, and in genetic engineering
• AFLP uses restriction enzymes to digest genomic DNA
• Ligation of adaptors to the sticky ends of the restriction
fragments
• A subset of the restriction fragments is then selected to be
amplified
• This selection is achieved by using primers complementary to the
adaptor sequence, the restriction site sequence and a few
nucleotides inside the restriction site fragments
• The amplified fragments are separated and visualized on
denaturing PAGE
• Autoradiography or fluorescence methodologies used for
identification
• Sequencing could also be used for identification

9. AFLP
• The AFLP technology has the capability to detect
various polymorphisms in different genomic
regions simultaneously
• Highly sensitive and reproducible
• ALP is widely used for the identification of genetic
variation in strains or closely related species of
plants, fungi, animals, and bacteria
• Also used in criminal and paternity tests
• To determine slight differences within populations
• Linkage studies to generate maps for quantitative
trait locus (QTL) analysis.

10. Advantages of AFLP
• AFLP has higher reproducibility, resolution, and
sensitivity at the whole genome level compared to
other techniques –RAPD, RFLP AND microsatelite
• Has the capability to amplify between 50 and 100
fragments at one time
• No prior sequence information is needed for
amplification
• AFLP is extremely beneficial in the study of taxa
including bacteria, fungi, and plants, where much is
still unknown about the genomic makeup of various
organisms.