Explains the Principle, Methodology and Application of RFLP

1. Dr. Deepa M.A
Assistant Professor of Botany
Government Arts College (Autonomous)
Coimbatore – 641 018
https://www.youtube.com/channel/UCXqGlEjNm
KrTmWVqMIngbDA

2. Principles
Mutations, variation, recombination’s may bring about changes in genomic DNA
sequences.
These changes in DNA sequences may generate or abolish or alter the position of
recognition site for restriction endonucleases.
https://commons.wikimedia.org/wiki/File:RFLP-figure.svg

3. If genomic DNAs are
1. digested with restriction enzyme,
2. electrophoresed,
3. blotted on membrane and
4. detected with labelled probes,
then polymorphism in the hybridization pattern will be revealed due to
change in restriction cleavage site.
Such variation is termed as’ Restriction Fragment Length
Polymorphism’ and is very useful to study changes in genomic DNA
sequences.

4. In eukaryotic DNA, cutting by restriction enzyme has no
biological significance and is only a chance occurrence of
appropriate sequence of nucleotides.
Such restriction sites are generally in regions of DNA that do
not code for any protein.

5. Procedure:
Isolate DNA from different plant species
Add restriction enzymes and digest the DNA to get fragments
Use same restriction enzyme for all the plants
After digestion load the DNA samples in the gel in the
electrophoresis unit and run the unit for about 2 hours
Once the separation is over capture the image of the DNA bands
obtained on the gel using Gel Doc system.
Analyze the variation in the pattern of DNA bands if present.

6. Practical applications of RFLP in selecting elite plant species:
1. RFLP analysis is highly discriminatory and species varying in just
one nucleotide can also be distinguished (provided the variation in their
restriction sites). It can be an important tool in identifying somaclonal
variation and selecting the clone having desirable characters.
HindIII Haemophilus influenzae
5'---AAGCTT---3'
3'---TTCGAA---5'
5'---AACCTT---3'
3'---TTCCAA---5'

7. 2. Genetic level disorders and defects can also be easily identified at
the cell level or plantlet level also. Because defective gene will give
rise to a different pattern if change is in the recognition site. So
RFLP identification of pathological conditions can also be identified.

8. https://commons.wikimedia.org/wiki/File:DNA_Profiling_-
_RFLP_Analysis.svg

9. Thus the plants carrying disease in the genetic level and acquired disease
characters or abnormality during developments can be easily detected.
Thus RFLP finds its application in plant biotechnology in various ways

10. To select phenotype having desired economic trait for selection in plant
breeding.
To identify the breeding lines and varieties
To characterize germplasm resources.
Use of genotypic specific RFLP patters can be done to distinguish
varieties of crops like maize, rice, soybean, oil seed, potato, etc.
To assess genetic diversity
For cytogenetic studies in plants.