RFLP ((Restriction Fragment Length Polymorphism)

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

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

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

Dominant vs Co-dominant B C A B C A B C A

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

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

Southern Blotting

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

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

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)

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)

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

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

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

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

RFLP autoradiogram.

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