RFLP & RAPD

A
SEMINAR ON
MOLECULAR MARKERS
RFLP-RAPD
DR. ARUNIMA KARKUN(ASST. PROF).
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 INRODUCTION
 DEFINITION
 HISTORY
 ANALYSIS
 TECHNIQUES
 ADVANTAGES
 DISADVANTAGES
 APPLICATIONS
 CONCLUSION
 REFERENCE
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MOLECULAR MARKER RFLP & RAPD
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 RFLP - RESTRICTION FRAGMENT LENGTH POLYMORPHISM
 RFLP & is very useful study in Genomic DNA Sequence.
 RFLP has been developed for chromosomes mapping of humans, mice, maize,
tomato,rice,etc.
 RFLP is a non-PCR based method .
 RFLP based Genetic Marker.
 In this Method DNA is digested with restriction Enzymes.
 RFLP is the co dominant marker.
 RFLP is 1-10 loci detected.
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 “The Variations in the Restriction DNA Fragments length between
individuals of a species is called RFLP.”
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 BOTSTEIN.et. al.(1980)-The DNA sequence variation Detected by this
method was termed RFLP.
 GRODZICKER first Described this phenomenon for mutant strain of
adenoviruses.
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Genomic DNA isolation.
Digestion with Restriction enzyme.
Fractions on a Agarosegel.
DNA in millions of restriction fragments fractionated in the gel
by Molecular marker weight .
DNA transferred out of the Gel on to a membrane filter southern
Hybridization.
Autoradiography
RFLP pattern with positive Bands.
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 Results are based on reliable genotypic characteristics' rather than on
phenotypes.
 RFLP based Genetic Marker
 RFLP is the co dominant marker
 RFLP & is very useful study in Genomic DNA Sequence
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 Time consuming.
 Multistep procedure.
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 SAMPLE– COLLECTIONS
 Tissue are cells of individual are collected to extract their DNA.
 The sample are collected separately.
 ISOLATION OF DNA
 DNA of the collected samples is isolated separately by standard
procedure& purified to get genomic DNA.
 RESTRICTION – DIGESTION
 Genomic DNA of each samples is cut with a restriction enzyme separately
To generate variable leanth of DNA fragments.
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 Restriction enzyme such as ECOR1, HINDIII ,Pst, etc ,are of much use for
RFLP.
 ELECTROPHORESIS
 The Digested genomic DNA of the samples are loaded into separate cells in
agarose or polyacrelamide gel &subjected to electrophoresis.
 BLOTTING OF DNA
 As in southern blotting the DNA is transferred to a nitrocellulose filter by
using a blotting set up.
 AUTOREDIOGRAPHY
 Images of radioactive probes are captured on an x- Ray film using
autoradiography.
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 Method involves:
 a) digestion of extracted DNA by restriction enzymes,.
 b) gel electrophoresis of fragments,.
 c) southern blot by specific probes and detection of specific sequences.
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 RFLP technique can be used in Forensic Medicines.
 To characterize Germplasm resources .
 Use of genotype specific RFLP patterns can be done to distinguish verities
in crop like maize, rice, soya bean, potato etc.
 To assess genetic diversity.
 To identify Breeding lines & varieties.
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 RAPD –Random Amplified Polymorphic DNA.
 RAPD are of much use to construct genetic maps.
 The RAPD is a PCR based Method.
 RAPD is a very general method for obtaining a molecular fingerprinting or
a strain species .
 RAPD analysis is a PCR based molecular marker technique.
 It is a Dominant marker.
 It s a 1-3 loci detected.
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 DEFINITION
 “The set of DNA generated by the Random PCR is called RAPD.”
 “If primers with arbitrary sequences are used for amplification, DNA
segments to be amplified will be selected at random and this provides
truly random sample of DNA markers and so is described as random
amplified polymorphic DNA (RAPD)”.
 HISTORY
 The method was Developed by J.G.K.Williams et.al in (1991).
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Genomic DNA Isolation / Cells or tissues .
Keep the tubes in PCR thermo cycler.
Denature DNA
940C 1 minutes
DNA Strands separated.
Annealing of primers.
Complementary DNA Synthesis (35 -45 cycles).
Amplified product separated by Gel Electrophoresis
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 Method involves:
a) extraction of DNA,
b) amplification by PCR using random primers.
c) gel electrophoresis of amplified DNA and visualization of markers.
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 RAPD is a Quick Method.
 RAPD Analysis can be performed in crude DNA Sample also.
 It detect Dominant variation in the Genome.
 It involves non-radioactive assays.
 RAPD required only a small amount of DNA samples.
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 RAPD is used as Genetic markers for constructing genetic maps of
higher organisms.
 Example – pines, rice etc.
 RAPD analysis help as to identify genes of high economic values
through comparison of RAPD fragments .
 Example- rice, wheat, maize , pea etc.
 RAPD Markers help to determine specific genes in chromosomes.
 RAPD can be used for identification of somatic Hybrid among the
developing regenerates .
 RAPD can be used for evaluation &character of genomic resources .
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RFLP RAPD
1-3 loci detected.  1-10 loci detected
Can detect allelic variants. Cannot detect allelic variants
Technique comparatively more
reliable.
Technique comparatively less
reliable.
Large quantity of purified DNA
required i.e. 2-10µg.
Quantity of DNA required for
analysis is small 10-50µg.
Different species specific probes are
required.
Same primers with arbitrary sequence
can be used for different species.
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 RFLP - RESTRICTION FRAGMENT LENGTH POLYMORPHIS
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RAPD –RANDOM AMPLIFIED POLYMORPHIC DNA.
RFLP is a non-PCR based method .
RAPD is a Quick Method.
RAPD required only a small amount of DNA samples.
RFLP based Genetic Marker.
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AUTHER NAME EDITION BOOK NAME
H.S.CHAWLA 1998 BIOTECNOLOGY
S.N.JOGDAND 2009 BIOTECNOLOGY
B.D.SINGH 2010 BIOTECNOLOGY
V.KUMARESAN 2011 BIOTECNOLOGY
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RFLP & RAPD

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