molecular markers principle and its applications

1. MOLECULAR MARKERS- RFLP,
RADP, AFLP
Done by,
P. MEENALOKSHINI
II MSC Biochemistry

3. BASIC PRINCIPLE

4.  The term Restriction Fragment Length Polymorphism, or
RFLP refers to a difference between two or more
samples of homologous DNA molecules arising from
differing locations of restriction sites, and to a related
laboratory technique by which these segments can be
distinguished.
 It is an genetic variation examined by cleaving the DNA
into fragments with restriction enzymes.
RFLP

5. Variations resulting in RFLP
1. SNPs(single base changes in the nucleotide
sequences)
2. Tandem repeats (VNTR)
3. Polymorphisms
4. Mutations

6.  The technique for detecting RFLPs involves
fragmenting the DNA sample by digestion with
restriction enzyme. The enzyme should be capable of
recognizing & cleaving DNA at specific short
sequences.
The fragments are separated by agarose gel
electrophoresis to get fragment of different length.
These are then transferred to a membrane via
southern blotting experiment.
RFLP-PRINCIPLE

7. Hybridization of the membrane to a labelled DNA
probe takes place.
Hybridization occurs between a DNA fragment of
specific length & complementary probe.
Thus, RFLP occurs when the length of a detected
fragment varies between individuals.

8.  Two DNA molecules from two plants (A and B) taken.
In plant A, a mutations has occurred leading to the
loss of restriction site that can be digested by EcoRI.
 The result is that when the DNA molecules are
digested by the enzyme Hindlll, there is no difference
in the DNA fragments separated. However, with the
enzyme EcoRI, plant A DNA molecules is not digested
while plant B DNA molecule is digested. This results in
a polymorphic pattern of separation.
Example

10. Isolation of DNA
Restriction digestion
Agarose gel electrophoresis
Transfer of DNA fragments to a membrane
(southern blot).
Hybridization of radioactive probe to
Specific fragment DNA
Membrane wash to remove excess probe.
auto radiography
Comparison of DNA pattern with known subjects.
RFLP Procedure

12.  Genetic disease analysis
 Genome mapping
 Forensic science
 Paternity determination
 Characterization of genetic diversity
 Characterization of breeding patterns in animal
population
APPLICATIONS

13.  Random Amplification of Polymorphic DNA is a type
of PCR reaction where the DNA amplified is random.
It is amplification of set of randomly distributed loci in
any genome using PCR with random primers.
RADP

14.  RAPD amplifies nanogram of genomic DNA under
lower annealing temperature by PCR. For this
synthetic oligonucleotides of random sequences are
necessary as primers.
 The product of amplification are generally separated
by agarose gel electrophoresis followed by staining
with ethidium bromide.
RADP- Principle

15. RAPD

16. 1. PCR
 Denature DNA
 94 ˚C - 1minute
 DNA strand separated .
 Annealing of primers
 Complementary DNA
synthesis(35-45 cycles)
2. Amplified product separated
by gel electrophoresis.
3. Staining with Ethidium
bromide.
RADP procedure

17. • Characterization, estimation of genetic relatedness and
determination of genetic diversity,Plant and animal breeding.
Provides means of identification of plant variety by a cultivator
& thus help in development of specific molecule identification.
• Able to distinguish between genotypes but limited to
comparisons of populations from a few sources – identification
marker.
• Genetic mapping – drawback is dominant so need more
character.
• Population and evolutionary genetics.
RAPD Applications

18. AFLP is a PCR based technique in which enzymes are
used to cut genomic DNA & adaptors are ligated to the
sticky ends of the restriction fragments. A subset of
restriction fragments is then amplified . It is developed
by KEYGENE in 1990’s.
AFLP

19.  The total cellular DNA is digested with restriction
enzymes & sticky ends are ligated with adaptors.
 Some of the fragments are selectively amplified with
2 PCR primers. The primers should have
corresponding adaptors & restriction site specific
sequence.
 The separation of amplicons is done by
electrophoresis. Band patterns are either visualized
by autoradiography or fluorescence methodologies.
AFLP Principle

20.  cDNA- AFLP , quantifying difference in gene
expression levels.
 TE AFLP, detection of transposable elements mobility.
Variations of AFLP

21. 1. Digestion with MseI and EcoRI
2. Ligation with adaptors complement to MseI and EcoRI cut
ends.
3. Amplification
Preselective amplification
Selective amplification
4. selected fragments are amplified and separated by agarose gel
electrophoresis.
AFLP Procedure

23. 1. Monitoring inheritance of agronomic traits
2. Diagnostic in genetically inherited disease
3. Pedigree analysis
4. Forensic typing - Parentage analysis
5. Identifying hybrids
6. Species level relationship
7. Also in some case at higher level relationship
AFLP- Applications

24. Thank you