1. RFLP ((Restriction Fragment Length Polymorphism)

2. RFLPs Restriction fragment length polymorphism Co-dominant
Requires: single copy DNA probe Restriction enzyme Southern blotting DNA polymorphism
RFLPs

3. A single nucleotide change can make a difference
AGATCT
Wild-type allele
TCTAGA
Restriction site
Mutant allele
AGAGCT
TCTCGA
Not a restriction site

4. Dominant vs Co-dominant
Most organisms we study are diploid
Two sets of chromosomes
Co-dominant:
the marker on both chromosomes is visible and distinguishable
Dominant: the marker is present and you can not see whether is coming from both chromosomes or from only one
Dominant vs Co-dominant

5. Dominant vs Co-dominantB C A B C A B C A

6. Differences in DNA-sequence between the two parents ( due to mutations )
Differences in restriction - enzym sites
RFLP-determination

7. The laboratory steps involved in RFLP detection
Isolation of DNA
Restriction digestion and gel electrophoresis
DNA transfer by Southern blotting
DNA hybridisation
The laboratory steps involved in RFLP detection

8. Southern Blotting

9. Blunt-ends Type
Restriction sites (I)
Sticky-ends Type

10. Restriction sites (II)
GAAATC
CTTTAG
No EcoRI site A B C
Parent 1 A D E C
Parent 2
GAATTC
CTTAAG
EcoRI site

11. Restriction sites (III)
Eco RI recognizes sequence of 6 basepairs
Theoretically every 4096 bases a Eco RI restriction site
Haploid tomato has about 2 x 109 basepairs
106 restriction sites per enzyme
Restriction sites (III)

12. Restriction sites (II)B C A D E
Parent 2
Parent 1
probe
Probe recognizes complementary sequence
Probe has a color label or is radio-active
Restriction sites (II)

13. Separation with gel electrophoresis; smaller fragments run fasterB C A D E
Parent 2
Parent 1
probe A C D E B
Separation with gel electrophoresis;
smaller fragments run faster

14. After Southern Blot with specific probeD E
Parent 2
Parent 1
probe A C D E B
After Southern Blot with specific probe

15. Production probes Isolation total DNA
Digestion with restriction enzymes
tomato Eco RI 106 fragments
Cloning and transformation plasmids to bacteria
Isolation individual bacteria + plasmids
Isolation vector DNA
Use as probe
Production probes

16. Sources of probes Nuclear DNA: - Genomic libraries - cDNA
Cytoplasmic DNA
- mitochondrial and chloroplast DNA
libraries
Repetitive sequences or minisatellite-type
Sources of probes

17. RFLP autoradiogram.

23. Other Applications of RFLP
Genetic diversity
Genetic relationships
History of domestication
Origin and evolution of species
Genetic drift and selection
Whole genome and comparative mapping
Gene tagging
Unlocking valuable genes from wild species
Construction of exotic libraries
Other Applications of RFLP