RFLP

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