Principle, working, application

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2. RFLP
Definition
• The variation in the restriction DNA fragment lengths
between individuals of a species is called restriction
fragment length polymorphism (RFLP).
• Laboratory technique to analyze and compare DNAs of
two or more individuals of a species or of different
species.
• Genetic structure of all the individuals of a species is
same, but at DNA level there are so many single base
variations between the individuals due to point mutation.
• If a single base is altered due to mutation restriction
enzymes never cut at the target sites.
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3. HISTORY
• Shortly after Kary Mullis invented the Polymerase Chain
Reaction (PCR) it was realized that short primers would
bind to several locations in a genome and thus could
produce multiple fragments.
• Williams et al. (1990) developed Random Amplified
Polymorphic DNA (RAPD) a technique using very short
10 base primers to generate random fragments from
template DNAs.
• RAPD fragments can be separated and used as genetic
markers or a kind of DNA fingerprint.
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4. RFLPTECHNIQUES
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5. The RFLPinvolves the following steps:
Sample collection
Isolation of DNA
Restriction digestion
Electrophoresis
Blotting of DNA
Making genomic DNA probes
Nucleic acid hybridization
Autoradiography
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6. Sample collection
Tissues or cells of individuals are collected to extract their
DNA. The samples are collected separately.
Isolation of DNA
Preparation of cell extract
lysozyme - digestion of polymeric compounds
EDTA - it removes Mg+ in the cell envelope
SDS - removes the lipids of the cell wall.
Purification of DNA
DNA isolation
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7. Restriction digestion
• Genomic DNA of each sample is cut with a restriction
enzyme separately to generate variable lengths of DNA
fragments.
• Restriction enzyme such as E.coR1, Hind ІІІ, Pst1.
• The restriction digestion is divided into two half is used for
DNA detection and the other is used for probe making.
.
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8. Electrophoresis
• The digested genomic DNA of all the samples are loaded into
separate wells in Agarose or Polyacrylamide gel and are
subjected to electrophoresis.
• The DNA fragments get
separated according
to their size by using
molecular weight
markers
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9. Blotting of DNA
Southern blotting
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10. • Each sample is electrophoresed to separate the DNA
fragments.
• Fragments of 0.5-2 kb are extracted from the gel and cloned
in pUC21 vector to construct rDNAs.
• These rDNAs are amplified by introducing them into
bacterial host and the amplified rDNAs are reisolated.
• Restriction digestion & electrophoresis.
• Radiolabelling by Nick translation or End labelling
• Genomic probes are formed.
Making genomic DNA probes
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11. Nucleic acid hybridization
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12. Autoradiography
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13. RFLPs are considered as the first class genetic markers to
construct high resolution linkage maps of chromosomes.
Used to construct chromosome map of human being, rice,
wheat, maize, and microbes.
Single gene diseases in man, plants and animals can be
identified with RFLP markers.
Monitoring inheritance of agronomic traits
Diagnostic in genetically inherited disease
Pedigree analysis,
Forensic typing - Parentage analysis
Identifying hybrids
Species level relationship
 Also in some case at higher level relationship
Applications of RFLP
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14. Advantages
Fingerprinting technique replacing RFLP
Highly polymorphic
High reproducibility
Identify through absent or present of fragment
Characters can be increased by changing the RE and
nucleotide at selective primers
Codominantly inherited.
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15. Disadvantages
Dominant – lose the codominant character
Homology – ability to differentiate different fragment with similar
size
 Mutation rate – high homoplasy
– High levels of variation - similarity between two taxa are low, so
both character and distance measures and tree reconstruction
programmes are increasingly inaccurate
– if levels of variation are high - Homoplasy
 Scoring - bias
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16. Definition
• The RAPD is a PCR based method to detect variations
between individuals of a species by selective amplification of
some polymorphic sequences in their genomes.
• Developed by J.G.K.Williams et.al. in 1991.
• Only least number of DNA fragments are considered for
RAPD analysis.
• The set of DNAs generated by the random PCR is called
RAPD.
• The RAPDs of one individual is differ from the RAPDs of
other individuals both in number and size of DNA fragments.
RAPD
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17. Components of a PCR and RAPD Reactions
PCR
1. Buffer (containing Mg++)
2. Template DNA
3. 2 Primers that flank the
fragment of DNA to be
amplified
4. dNTPs
5. Taq DNA Polymerase (or
another thermally stable DNA
polymerase)
RAPD
1. Buffer (containing Mg++) -
usually high Mg++
concentrations are used lowering
annealing stringency
2. Template DNA
3. 1 short primer (10 bases) not
known to anneal to any specific part
of the template DNA
4. dNTPs
5. Taq DNA Polymerase (or
another thermally stable DNA
polymerase)
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18. Major steps in RAPD analysis
• Sample collection
• DNA isolation
• PCR
Each genomic DNA is separately treated with Taq DNA
polymerase, a primer, dATP ,dTTP, dGTP, dCTP, and
polymerization buffer.
All these reaction mixture are kept separately in a PCR
equipment.
subjected t o denaturation – 94°c for 1minute
primer annealing -36°c for 2minutes
polymerization – 72°c for 1.5minutes.
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19. • During the primer annealing, the primer molecules get bind with the
polymorphic sequences found here and there throughout the genome by
complimentary base pairing.
RAPD Method
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20. • Provides 3’-OH for synthesis of new strands by polymerase
activity during polymerization reaction.
• As a result of PCR amplification, each tube containing RAPD
fragments.
• DNA fragments has a flanking primer at its 5’ end.
• Electrophoresis.
• Examined under the
UV light illuminator
and photographed.
• Light bands of the
markers shows the
molecular weights
of each individual in
different lanes
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21. Uses of RAPD
• RAPDs are used as genetic markers for constructing genetic maps of
higher organisms.
• Helps to identify genes of high economic value through comparison
of RAPD fragments.
• Distinguishes one individuals from others of a species as
fingerprinting analysis
• Determine specific genes in chromosomes
It has become widely used in the study of
• Genetic diversity/polymorphism,
• Germplasm characterization,
• Genetic structure of populations,
• Domestication,
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22. • Detection of somoclonal variation,
• Cultivar identification,
• Hybrid purity,
• Genome mapping,
• Developing genetic markers linked to a trait in question,
• Population and evolutionary genetics,
• Plant and animal breeding,
• Animal-plant-microbe interactions,
• Pesticide/Herbicide resistance,
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23. Advantages of RAPD
• No need for species specific probes in RAPD
• Probes prepared for one species may also used for other
species
• Quick method
• Can be performed in crude DNA samples also
• Requires only a small amount of samples
• Doesn’t require radioactive probes and hybridization.
• It detects dominant variations in the genome
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24. Disadvantages
• Profiling is dependent on the reaction conditions
• Profiles are not able to distinguish heterozygous from
homozygous individuals- dominant marker
• Lack of a prior knowledge on the identity of the amplification
products.
• Problems with reproducibility (sensitive to changes in the
quality of DNA, PCR components and PCR conditions).
• Problems of co-migration (do equal-sized bands correspond to
the same homologous DNA fragment). Gel electrophoresis can
separate DNA quantitatively, cannot separate equal-sized
fragments qualitatively (i.e. according to base sequence).
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25. References
• N.Senthikumar & G.Gurusubramanian, Random amplified
polymorphic DNA (RAPD) markers and its applications 2011,
www.sciencevision.in
• Timothy G. Standish, Ph. D. , Random Amplified Polymorphic
DNA , (ppt).
• M.O. Dayhoff and R.V. ECK, Molecular techniques,(ppt).
• Biotechnology, V.Kumaresan, Saras publications.
• Biotechnology, U.Sathyanarayana.
• Gene Biotechnology, S.Jogdand.
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