This document summarizes the random amplified polymorphic DNA (RAPD) technique. RAPD is a type of PCR that uses random nucleotide primers to amplify unknown regions of genomic DNA. It was developed in 1991 and involves using single, short random primers to amplify random DNA segments. The amplified products are then separated via gel electrophoresis and visualized. RAPD is a quick and inexpensive molecular marker technique that requires no prior DNA sequence knowledge, but lacks reproducibility and produces dominant markers. Its applications include assessing genetic diversity, mapping genomes, and use in breeding and evolutionary studies.

1. Semester-4
Subject: Biotechnology
Topic: RAPD
Presented by:
R. petchiammal
II PG Microbiology
Reg no: 20201232516112
Submitted to:
Dr. G. Ramanathan
Assistant professor
Department of
Microbiology
SPKC- Alwarkurichi

2.  Marker
 Definition
 History
 Principle
 How RAPD works?
 Molecular marker
 RAPD- Procedure
 Protocol
 Advantages
 Disadvantages
 Applications
 RAPD-RFLP-AFLP- comparision
 References
SYNOPSIS

3. MARKER:
 Any genetic trait that can be identified with
confidence and relative case and can be followed in a
mapping population is called as Genetic marker.
 There are three types of markers namely:-
Morphological marker
Biochemical marker
Molecular marker

4. DEFINITION:
 RAPD that is defined by differences between
individuals in terms of DNA regions either being / not
being amplified in a polymerase chain reaction primed
by random oligonucleotides sequences.
 It is a type of PCR reaction, but the segments of DNA
that are amplified are random.
 RAPD – is a lab technique used to amplify unknown
(random) DNA segments.

5. HISTORY:
 The method was Developed by J.G.K. Williams et.al
in (1991).
PRINCIPLE:
 RAPD analysis is a PCR based molecular marker
technique. Single short oligonucleotide primer is
arbitrarily selected to amplify a set of DNA
segments distributed randomly throughout the
genome.

6. How RAPD works?

7. MOLECULAR MARKER:
 Molecular marker is a gene / DNA sequence with a
known location on a chromosome that can be used to
identify cells.
 A marker must be polymorphic that it must exist in
different forms so that chromosome carrying the
mutant gene can be distinguished from the
chromosome with the normal gene by marker.

8. PROCEDURE:
RAPD involves following steps:-
 The DNA of a selected species is isolated.
 An excess of selected decaoligonucleotides added.
 This mixture is kept in a PCR equipment and is
subjected to repeated cycles of DNA denaturation –
renaturation – DNA replication.
 During this process, the decaoligonucleotide will pair
with the homologous sequence present at different
locations in the DNA.

9. DNA replication extend the decaoligonucleotide and
copy the sequence continuous with the sequence with
which the selected oligonucleotide has paired.
 The repeated cycles of denaturation- renaturation-
DNA replication will amplify this sequence of DNA.
 Amplifications will takes place only of those regions of
the genome that has the sequence complementary to
the decaoligonucleotides at their both ends.
 After several cycles of amplification the DNA is
subjected to gel electrophoresis.
 The amplified DNA will form a distinct band. It is
detected by ethidium bromide staining & visible
fluorescence’s under U.V. light.

10. PROTOCOL:
Isolation of DNA
Keep the tubes in PCR thermocycler
Denature the DNA (94 c,1min)
DNA strands separated
Decaoligonucleotides enzyme,
primer, Taq DNA polymerase
Annealing of primer(36 c, 2min)
Primer annealed to template DNA strands
DNA synthesis (72 c, 1.5min)

11. Complementary strand synthesis
35 to 45 cycles
Amplified products separated by gel electrophoresis
Bands detected by Ethidium bromide staining

14. ADVANTAGES:
 It requires no DNA and sequence information for the
design of specific primers.
 It involves no blotting / hybridisation steps, hence, it is
quick, simple and efficient.
 High number of fragments.
 Arbitrary primers are easily purchased.
 Unit costs per assay are low compares to other marker
technologies.

15. DISADVANTAGES:
 Nearly all RAPD markers are dominant.
 Lack of a prior knowledge on the identity of the
amplification products.
 Problems with reproducibility.
 Problems of co-migration.

16. APPLICATION:
 Genetic diversity
 Genetic structure of poopulations
 Genome mapping
 Plant and animal breeding
 Pesticide/ herbicide resistence
 Hybrid purity
 Populations and evolutionary genetics