This document discusses restriction fragment length polymorphism (RFLP), a technique used for DNA fragment separation and identification. It outlines the principles, procedural steps, applications, advantages, and disadvantages of RFLP, emphasizing its roles in genome mapping, genetic disease analysis, and criminal investigations. Key considerations include the need for high-quality DNA, the complexity and labor intensity of the methodology, and the high costs associated with its use.

1. MOLECULAR MARKERS IN GENOME ANALYSIS
R F L P
RESTRICTION FRAGMENT LENGTH POLYMORPHISM
BY: RITESWARI NAIK
M.SC ,ZOO 2ND YEAR

2. CONTENT
➢ 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. PRINCIPLE OF RFLP
➢ 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. PATTERN GENERATED DEPENDS MAINLY
ON
➢ 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. CONSIDERATIONS FOR USE OF Rflp
➢ 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. RESTRICTION ENZYMES
➢ Endonuclease that cleaves the double-stranded DNA at specific 4-8 nucleotide long palindromic sequence.

7. PROCEDURES OR STEPS OF RFLP TEST
❖ Step 1: Collection of sample DNA is extracted from the available tissue sample.
To extract DNA from Samples like

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

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.

12. APPLICATION OF RFLP TEST
❑ Genome mapping
❑ Genetic disease analysis

13. ❑ To detect mutated gene

14. ❑ Criminal investigations

15. ❑ Paternity test

16. ADVANTAGES
• 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. DISADVANTAGES
• 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).

18. REFERENCE
I. Alec Jeffreys: The English scientist invented RFLP in 1984 while researching hereditary diseases.
II. Southern EMn 1975, Southern EM published research on detecting specific sequences in DNA fragments
separated by gel electrophoresis.
III. Donis-Keller H, Green P, Helms C, Cartinhour S, Weiffenbach B, Stephens K, Keith TP, Bowden DW, Smith DR,
Lander ES et al. In 1987, Donis-Keller et al. published research on a genetic linkage map of the human genome.
IV. Evola SV, Burr FA, Burr B: in 1986, Evola et al. published research on the suitability of RFLP as genetic markers in
maize.
V. Eiadthong et al.: In 1999, Eiadthong et al. published research on classifying 13 Mangifera species using a
combination of RFLP and amplified fragment length polymorphism (AFLP).
VI. Park and Nishikawa : In 2012, Park and Nishikawa published research on developing molecular techniques based
on PCR-RFLP to identify two cultivated grain species.

19. THANK YOU