This document discusses the RFLP (restriction fragment length polymorphism) technology. It describes the key steps in RFLP which are isolating DNA from samples, digesting the DNA with restriction enzymes, separating fragments via gel electrophoresis, transferring DNA to membranes, and detecting bands through hybridization with probes. The document outlines the equipment needed and discusses advantages like being robust and able to estimate heterozygosity, as well as disadvantages like being time-consuming and requiring large DNA amounts. Finally, it presents applications of RFLP in areas like genetic diversity analysis and gene mapping.

1. Jitendra Kumar
College of Fisheries, Mangalore

2.  RFLP technology
• Isolating DNA
• Restriction digestion and gel electrophoresis
• DNA transfer by southern blotting
 DNA hybridigestion
 Equipment
 RFLP technology in pictures
 Interpreting RFLP bands
 Advantage and disadvantage of RFLPs

3. RFLP detection relies on the possibility of comparing
band profiles generated after restriction enzymes
digestion of target DNA.
 The laboratory steps involved are as follows:
 Isolation of DNA
 Restriction digestion and gel electrophoresis
 DNA transfer by southern blotting

4.  DNA hybridization
• The procedure
• The DNA probe
• Sources of probes
 Equipments

5.  Total DNA is extracted from fish cells
 DNA must be clean and of high molecular weight
 Complication:
• Breaking during isolation
• DNA degraded by nucleases
• Isolation of secondary fish metabolites

9.  Nuclear DNA
• Genomic libraries
• cDNA
 Cytoplasmic DNA
 The species specificity of many single-locus probes
requires that libraries be built when studying new
species. However, probes from related genera can be
often be used.

10.  Repetitive sequence or minisatellite-type:
• Basic motif of 10 to 60bp in tendem
• Highly variable between human individuals
• Polymorphism in the no. of repeated units (also called VNTRs)
 In fish, probes from an internal repeat from the protein lll
gene of the bacteriophase M13 have been to reveal
minisatellite sequences

11.  Resource:
• Distilled and/or deionised water
• Reagents
 Equipment:
• Refrigerator and freezer
• Laminar flow hood
• Centrifuge
• Power supply units
• Hot plate or microwave

12. • pH meter
• Standard balance
• Gel electrophoresis units
• Dark room
• Uv transilluminatior

20.  Highly robust methology with good transferability between
laboratories
 Co-dominantly inherited and as such can estimate
heterozygosity
 No sequence information required
 Because based o sequence homology, highly recommended
for phylogenetic analysis between related species
 Well suited for constructing genetic linkage maps
 Locus-specific marker, which allow synteny studies

21.  Discriminator power can be at species and/or population
levels (single-locus probes), or individuals level (multi-
locus probes)
 Simplicity- given the availability of suitable probes, the
techniques can readily be applied to any plant

22.  Large amounts of DNA required
 Automation not possible
 Low level of polymorphuism in some species
 Few loci detected per assay
 Need a suitable probe library
 Time consuming, especially with single copy probes
 Costly
 Distribution of probes to collaborating laboratories
required

23.  Moderately demanding technically
 Different probes/ enzymes combinations may be needed

24.  Genetic diversity
 Genetic relationships
 History of domestication
 Origin and evaluation of species
 Genetic drifts and selection
 Whole genome and comparative mapping
 Gene tagging
 Unlocking valuable genes from wild species
 Construction of exotic libraries

25.  The RFLP technology detects length changes in target
DNA molecules after restriction enzymes digestion
 RFLP bands are detected by hybridizing the target DNA
with a DNA probes
 RFLP banding pattern reflected different mutational
events at the hybridizations site of the probe or its
neighboring region
 RFLP is highly robust technology, but time consuming
and technically demanding