Download Restriction sites

RFLP ((Restriction Fragment
Length Polymorphism)
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
Wild-type allele
AGATCT
TCTAGA
Restriction site
Mutant allele
AGAGCT
TCTCGA
Not a restriction site
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 Codominant
B
C
A
A
B
C
A
B
C
Differences in DNA-sequence
between the two parents ( due to
mutations )
 Differences in restriction - enzym
sites

RFLP-determination
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
A
A
B
D
C
E
C
No EcoRI site
Parent 1
Parent 2
GAATTC
EcoRI site
CTTAAG
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)
A
B
C
Parent 1
probe
A
D
E
C
Parent 2
probe
Probe recognizes complementary sequence
Probe has a color label or is radio-active
Restriction sites (II)
A
B
C
Parent 1
probe
A
D
E
C
Parent 2
B
probe
E
Separation with gel
electrophoresis;
C
C
D
smaller fragments run faster
A
A
A
A
B
D
C
probe
E
C
Parent 1
Parent 2
B
probe
After Southern Blot with specific
probe
E
C
C
D
A
A
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

Nuclear DNA:
- Genomic libraries
- cDNA
Cytoplasmic DNA
- mitochondrial and chloroplast DNA
libraries
 Repetitive sequences or minisatellite-type

Sources of probes
RFLP autoradiogram.
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
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Other Applications of RFLP