Dr. Derakhshandeh, PhD

Mutation ScreeningMutation Screening

2

TYPE OF MUTATIONSTYPE OF MUTATIONS

WHICH TECHNIQUES DETECT WHAT TYPE OF MUTATIONS

In classical genetics, three types of mutations are distinguished:

3

Different types of Different types of mutationsmutations

genome mutations: changes in chromosome number

chromosome mutations: changes in chromosome structure

gene or point mutations: mutations where changes are at molecular level

4

genome genome mutations:mutations:

changes in changes in chromosome chromosome

numbernumber

5

TechniquesTechniques

Karyotyping, conventional cytogenetics

6

Down Syndrome Down Syndrome ((Trisomy 21(Trisomy 21(

Trisomy 2(Trisomy 2(

7

chromosome mutations: chromosome mutations: changes in chromosome changes in chromosome

structurestructure

8

CHANGES IN CHROMOSOME CHANGES IN CHROMOSOME STRUCTURESTRUCTURE

TranslocationsLarge Deletions/InsertionsInversionsDuplications/Amplifications

9

TechniquesTechniques

Conventional cytogenetics molecular cytogenetics

FISHMolecular:

PFGE, Southern blotting, Northern BlottingFluorescence Dosage analysis

large deletionsInsertionsduplications

10

Interphase FISH Examples

13 (green),13 (green), and 21 (red) and 21 (red)18 (aqua),18 (aqua), X (green),X (green), and Y (red). and Y (red).

11

gene or point gene or point mutations:mutations:

mutations where mutations where changes are at changes are at molecular levelmolecular level

12

Methods for Methods for detection of known detection of known

mutationsmutations

Methods for Methods for detection of unknown detection of unknown

mutationsmutations

13

Methods for detection of Methods for detection of known mutationsknown mutations

PCR and size separation eg. DMD PCR and restriction enzyme digestion eg. SMN

exon 7 & 8 Allele specific amplification (ASA)

Allele refactory mutation system (ARMS) eg. CF

Allele specific oligonucleotide hybridisation (ASO) Dot Blot eg. CF DNA chips eg. Brca1

Genomic DNA sequencing

14

ARMSARMS

Amplification Refractory Amplification Refractory Mutation System Mutation System

Allele Specific PCR (ASPCR) PCR Amplification of Specific Alleles (PASA)

15

UsesPopulation screening

rapid (1 working day) inexpensive non-isotopic

Used for testing for B-thalassaemia Cystic Fibrosis Sickle-cell anaemia Phenylketonuria Apolipoprotein E, etc

16

Modifications/Adaptions to the original ARMS methodology

Multiplex ARMS

17

To set up multiplex ARMS

Determine commonest mutations in the population

develop the muliplex ARMS these mutations

validate the results of the multiplex test on samples with another methology

18

Agarose gels showing the Agarose gels showing the feasibility of the ARMS feasibility of the ARMS conceptconcept

19

ARMS-PCRARMS-PCR

1 2 3 4 5 6 7 8 9 10 11 12 N M N M N M N M N M N M

20

Methods for detection Methods for detection of unknown of unknown mutationsmutations

21

Methods for unknown mutations (diagnostic methods)

These methods are relatively simple, but still require:

experience and skill to perform.

22

BRCABRCA

23

BRCA1 Gene

24

BRCA2 Gene

25

SSCP single strand conformation

polymorphism

simplicity clearly by heteroduplex analysis (HA)

26

Pedigree of a selected family with breast cancer

27

SSCP AnalysisBRCA1 Exon 15, 4650delCA

28

Pedigree of a selected family with breast cancer

29

SSCP AnalysisBRCA1, Exon 20,Nt 5382

30

SSCP AnalysisExon 11pi BRCA1 MS R1347G

31

A woman having amniocentesis