D. Y.

PATIL UNIVERSITY, NAVI MUMBAI

SCHOOL OF BIOTECHNOLOGY AND BIOINFORMATICS
PRACTICAL MANUAL PREPARATION
ProgrammePractical
Title-

M.Tech Integrated

Semester-

VI

Random Amplification of Polymorphic DNA.

Experiment. No.
Aim-

To understand the concept of DNA fingerprinting by Random

Amplification of Polymorphic DNA.

Principle-

A DNA fingerprint can be called a genetic photograph of an individual.

RAPD is a very general method for obtaining a molecular fingerprint of a
species or strain. Low stringency PCR amplification of genomic DNA
using a single short primer (10 to 22 mer) of arbitrary sequence to
generate a set of fragments that is characteristic of the variety from which
the DNA was isolated. Each fragment in the profile is the result of
hybridization and extension of a primer on opposite DNA strands in the
appropriate orientation and separated by such a distance allowing efficient
PCR.
This random primer is able to bind under appropriate conditions to a
number of partially or perfectly complementary sequences at unknown
locations in the genome of an organism. If the binding sites occur in a
spacing and orientation that allows amplification of DNA fragments,
fingerprinting patterns are produced.
By performing similar experiments with different primers and many
crops/varieties, quantitative data can be derived which can be then used to
prepare dendrograms for taxonomic studies. A particular band can also be
considered to be a Mendelian trait of an organism and can be used as a
molecular marker to study the segregation of other traits of economic
significance.

Requirment-

Genomic DNA samples (4 samples of rice DNA), Random primer, Taq

DNA polymerase, 10X assay buffer, 10mM dNTP mix, Nuclease free
water, 100 bp DNA ladder (ready to use), Gel loading buffer (store at
-20C )
50X TAE, agarose, PCR tubes (store at RT)
Miscellaneous:
Thermocycler, UV transilluminator, crushed ice, micropipettes, tips,
gloves

Procedure:
1.

2.

3.

Prepare a PCR cocktail for 4 reactions.

Add the following reagents to one PCR vial in the following order.
Nuclease free water
60.4L
10X assay buffer
10L
10mM dNTP mix
4L
Random primer
4L
Taq DNA polymerase
1.6 L
1 gently.
Mix the contents uniformly and
Note: all the above additions to be done on ice
Aliquot 20 L of above PCR cocktail to each of the four different PCR

D. Y. PATIL UNIVERSITY, NAVI MUMBAI

SCHOOL OF BIOTECHNOLOGY AND BIOINFORMATICS
PRACTICAL MANUAL PREPARATION
Programme-

Semester-

Practical
TitleExperiment. No.
Observation
Table
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