2. • Principle of RFLP
• Pattern generated depends mainly on
• Considerations for use of RFLPs
• Restriction enzymes
• Procedures or steps of RFLP test
• Application of RFLP test
• Advantages
• Disadvantages
3. RFLP is an enzymatic procedure for separation and
identification of desired fragments of DNA.
Using restriction endonuclease enzymes fragments of
DNA is obtained and the desired fragment is detected
by using restriction probes.
May be used to differentiated two organism by
analysis of patterns derived from cleavage of their
DNA.
4. Differences in DNAs of selected strains
Restriction enzymes used
DNA probe employed for southern hybridization
Point and frameshift mutations
Differences in alleles for a particular sequence
5. Relatively slow process.
Use of radioisotopes has limited RFLP use to
certified laboratories (but non-radioactive labeling
systems are now in wide use)
Co-dominant markers; often species-specific; highly
locus-specific
Specific to a single clone/restriction enzyme
combination
Need high quality DNA
Need to develop polymorphic probes
Expensive
6. • Endonuclease that cleaves the double-stranded DNA
at specific 4-8 nucleotide long palindromic
sequence.
Types
TYPE
1
TYPE
2
TYPE
3
7. • Step I: Collection of sample
DNA is extracted from the
available
tissue sample.
8. • Step II: Restriction digest
The DNA in each sample is digested with the
same restriction enzyme(s).
The enzyme RE has specific restriction site on
the DNA, so it cut DNA into fragments.
Different size of fragments are generated
along with the specific desired fragments.
9. • Step III: Gel electrophoresis
The digested fragment are run in polyacrylamide
gel electrophoresis or Agarose gel electrophoresis to
separate the fragments on the basis of length or size
or molecular weight.
• Step IV: Denaturation
The gel is placed in sodium hydroxide (NaOH)
solution for denaturation so that single stranded
DNA are formed.
10. • Step V: Blotting
The single stranded DNA obtained are transferred into charge
membrane i.e. Nitrocellulose paper by the process called capillary
blotting or electro-blotting.
• Step VI: Baking and blocking
The nitrocellulose paper transferred with DNA is fixed by
autoclaving.
Then the membrane is blocked by using bovine serum albumin or
casein to prevent binding of labeled probe nonspecifically to the
charged membrane.
11. • Step VI: Hybridization and visualization
The labeled RFLP probe is hybridized with DNA on the
nitrocellulose paper.
The RFLP probes are complimentary as well as labeled
with radioactive isotopes so they form color band
under visualization by autoradiography.
16. • Produces semi-dominant markers, allowing
determination of homozygosity, or heterozygosity.
• Stable and Reproducible, gives constant results over
time, and location.
• No prior information on DNA sequence is required.
• Relatively simple technique.
17. •Very long methodology before results are gained.
•High labour requirements.
•High quality, and large quantities of DNA must be used.
•Must frequently work with radio isotopes.
•Many probes are not available depending on species.
•Too many polymorphisms may be present for a short probe.
•Cost of development is very high due to time, and labour
requirements.
•Low frequency of desired polymorphisms in polyploid
plants (eg. Wheat).
