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

Wild-type allele

AGATCT 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
A
B C

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)

GAAATC

CTTTAG
A B C

No EcoRI site

Parent 1
Parent 2

GAATTC EcoRI site CTTAAG

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 (II)

A B probe A D E C C

Parent 1

Parent 2

probe

Probe recognizes complementary sequence Probe has a color label or is radio-active

B probe

Parent 1 Parent 2
B

E probe

Separation with gel electrophoresis;

C D

smaller fragments run faster

A

C probe E C probe

Parent 1 Parent 2
B

After Southern Blot with specific probe

E C C D

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

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