mutation detection systems navi

PREPARED BY

NAVEENA GIRISH

DEPARTMENT OF PLANT SCIENCE

CENTRAL UNIVERSITY OF KERALA

2 TYPES

VISIBLE SLCBIOCHEMICAL

SLC

WIDELY USEDUses 5 or 7 loci

Uses up to 20 enzymes

T O DETECT QUANTITATIVE MUTATION IN MAMMALIAN

GERMLINE CELLS

A visible specific locus mutation is a genetic change that

alters factors responsible for coat color and other visible

characteristics of certain mouse strains

principle

to cross individuals who differ with respect to the genes

present at certain specific loci, so that a genetic alteration

involving the standard gene at any one of these loci will

produce an offspring detectably different from the standard

heterozygote.

FEMALE

HOMOZY

GOUS

MALE +

TEST

REAGENT

PROGENY

SCREENING FOR

MUTATION

BIOCHEMICAL SPECIFIC LOCUS TEST

A biochemical specific locus mutation is a genetic change resulting from a DNA lesion causing alterations in proteins that can be detected by electrophoresis methods.

The principle of the MBSL is that heritable damage to the genome can be detected by electrophoresis analysis of proteins in the tissues of the progeny of mice treated with germ cell mutagens

Treated males are then mated to untreated females to produce F1 progeny. Both blood and kidney samples are taken from progeny for electrophoresis analysis. Up to 33 loci can be examined by starch-gel electrophoresis and broad-range isoelectric focusing. Mutants are identified by variations from the normal electrophoresis pattern. Presumed mutants are bred to confirm the genetic nature of the change.

Single generation & visual detection is used to identify

mutation – SO USEFUL

OFFSPRING EXAMINATION

BIRTH AND WEANING

TISSUE SAMPLING

ELECTROPHORESIS

MUTANT IDENTIFICATION

BLOOD -6 LOCI

KIDNEY – 27 LOCI

AMES TEST

Industrial chemicals potentially harmful

to humans

We can know it using lab animals

But it is time consuming and expensive

Bruce Ames 1974

PRINCIPLEDamage to DNA mutation Cancer

90% carcinogen = mutagenMutation in bacteria = carcinogenesis in humans

I used Salmonella typhimurium

with dna repair system

inactivated and defects in

lipopolysacharide

4 strains used

carcinogenic compounds in hair dyes removed by the help of

Ames test

One of the four strains detect base pair substitutions

Other three detect frameshift mutation

DROSOPHILA

MELANOGASTER

DEFINED MUTATION RATE

INTIAL EXPERIMENTS WAS

FAIL

BUT 1927 BERLIN

CONFERENCE HE SURPRISED

OTHERS

X- RAYS INDUCES

MUTATION

7 YEARS

BUT HE SAID NO QUALITATIVE DIFFERENCE BETWEEN SPONDANEOUS AND

INDUCED MUTATIONS

ClB METHOD – TO STUDY MUTATIONS HAPPENING ON X CHROMOSOME

He received Nobel prize in 1945

MULERS -5 METHOD

TO TEST X CHROMOSOME

The progeny of F2 generation indicate

whether or not lethal mutations occurred on

males x chromosomes

If lethal mutation occurred on wild type male

– it die

C – prevents crossing over & recombination

So lethality reside in one chromosome itself

C – prevents cross over

There is 2 markers

B – bar eye

Wa for apricot

recessive mutation can only express if

there is homozygosity

Attached x – to screen F1 mutations on x

of males

X^X – REPRESENTATION

Compound chromosome 2 x chromosome

attached

Inheritance as single unit

X^X female

ATTACHED X CHROMOSOME