MUTATION AND

DETECTION OF
MUTATION USING RFLP
(Restriction Fragment
Length Polymorphism)
By
Muhammad Irsan Saleh
Dept. of Pharmacology, Sriwijaya University

Mutations
Any change in the DNA sequence of an
organism.
Mutations are the source of the altered
versions of genes that provide the raw
material for evolution.
Only a small percentage of mutations
causes a visible but non-lethal change in the
phenotype.
Mutations are generally recessive
Mutations are rarely beneficial

Types of DNA Change
Base changes : one base is converted to another.
Transitions : one purine is changed to another purine
or one pyrimidine is changed to another pyrimidine
Transversions : a purine is substituted for a
pyrimidine, or a pyrimidine is substituted for a purine
Gain or loss of one or a few bases (insertion or deletion).
Insertion of whole new sequences, often due to
movements of transposable elements in the DNA.
Deletions of large segments of DNA also occurs.
Types of Mutation
Silent mutations (synonymous mutations):
base changes do not change the amino
acid sequence of the protein.
Missense mutations : substitute one amino
acid for another.
Nonsense mutations convert an amino
acid into a stop codon.
Sense mutations are the opposite of
nonsense mutations. Here, a stop codon
is converted into an amino acid codon.
Frameshifts and
Reversions
If a nucleotide or two is added or removed, the
groupings of codons is altered. This cause a
frameshift mutation, where the reading frame of the
ribosome is altered.
Frameshift mutations result in all amino acids
downstream from the mutation site being completely
different from wild type. These proteins are generally
non-functional.
A reversion is a second mutation that reverse the
effects of an initial mutation, bringing the phenotype
back to wild type (or almost).
Frameshift mutations sometimes have second site
reversions, where a second frameshift downstream
from the first frameshift reverses the effect.
Germinal vs. Somatic
Mutations
Mutations can occur in any cell. They
only affect future generations if they
occur in the cells that produce the
gametes: these are germinal or
germ line mutations.
Mutations in cells other than germ line
cells are somatic mutations.
RFLPs
Restriction fragment
length polymorphism
Co-dominant
Requires:
single copy DNA probe
Restriction enzyme
Southern blotting
DNA polymorphism
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
B C A
B C
A
B C A
Dominant vs Co-dominant
RFLP-determination
Differences in DNA-sequence between the
two parents ( due to mutations )
Differences in restriction - enzym sites
The laboratory steps
involved in RFLP detection
Isolation of DNA
Restriction digestion and gel
electrophoresis
DNA transfer by Southern blotting
DNA hybridisation

Southern Blotting
Restriction sites (I)
Blunt-ends Type
Sticky-ends Type
Restriction sites (II)
B C
A D E C
A
Parent 2
Parent 1
GAATTC
CTTAAG
GAAATC
CTTTAG
No EcoRI site
EcoRI site
Eco RI recognizes sequence of 6 basepairs

Theoretically every 4096 bases a Eco RI
restriction site

Haploid tomato has about 2 x 10
9
basepairs

10
6
restriction sites per enzyme
Restriction sites (III)
Restriction sites (II)
Probe recognizes complementary sequence
Probe has a color label or is radio-active

B C
A D E C
A
Parent 2
Parent 1
probe
probe
Separation with gel
electrophoresis;
smaller fragments run faster
A
C C
A
D
E
B
B C
A D E C
A
Parent 2
Parent 1
probe
probe
After Southern Blot with specific
probe
A
C C
A
D
E
B
B C
A D E C
A
Parent 2
Parent 1
probe
probe
Production probes
Isolation total DNA
Digestion with restriction enzymes
tomato Eco RI 10
6
fragments
Cloning and transformation plasmids to
bacteria
Isolation individual bacteria + plasmids
Isolation vector DNA
Use as probe

Sources of probes
Nuclear DNA:
- Genomic libraries
- cDNA

Cytoplasmic DNA
- mitochondrial and chloroplast DNA
libraries
Repetitive sequences or minisatellite-type

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