Mutagenesis and Genetic Screens

Fig. 12-39

General pathway for mutational dissection of a biological process

“Forward Genetics”

Fig. 12-39

General pathway for mutational dissection of a biological process

“Forward Genetics”

Classification of Mutant Type

Fig. 12-38

Test for type of mutation recovered: haplo-insufficient null

Fig. 12-38

Test for type of mutation recovered: haplo-insufficient null

Test for type of mutation recovered: haplo-insufficient leaky

Fig. 12-38

Fig. 12-38

Test for type of mutation recovered: gain-of-function mutation

Test for type of mutation recovered: neomorphic mutation

Fig. 12-38

Fig. 12-39

General pathway for mutational dissection of a biological process

“Forward Genetics”

From phenotype to gene• Once an interesting

mutant is found and characterized, we want to find the gene in which the mutant occurred

• Positional cloning– First use genetic

mapping– Then use chromosome

walking

chromosome contig candidate genes mutation

Candidate-gene approach• If the mutated gene is

localized to a sequenced region of the chromosome, then look for genes that could be involved in the process under study

• Last step: confirm gene identification– Rescue of phenotype– Mutations in same gene in

different alleles

Insertional mutagenesis• Alternative to chromosome walking

– To reduce time and effort required to identify mutant gene

• Insert piece of DNA that disrupts genes– Inserts randomly in chromosomes

• Make collection of individuals– Each with insertion in different place

• Screen collection for phenotypes• Use inserted DNA to identify mutated gene

Insertional mutagens

• Transposable elements– Mobile elements jump from introduced DNA

• e.g., P elements in Drosophila

– Or start with a small number of nonautonomous elements

– Mobilize by introducing active element• e.g., AC/DS elements in plants

• Single-insertion elements– e.g., T-DNA in plants

• Once insert, can’t move again

Genome-Wide Phenotypic Analysis:

“Phenomics”

High-Throughput Genetics

Applications of genomics approaches to genetics

High-throughput genetic screens

• Some genetic screens are relatively straightforward– e.g., For a visible phenotype like eye color

• If phenotype is subtle or needs to be measured, the screen is more time consuming– Examples

• Seed weight• Behavioral traits

Industrial setting for screens

2002 Paradigm Genetics, Inc. All rights reserved. Used with permission.

High-throughput genetic screen• Paradigm Genetics, Inc.

performs “phenotypic profiling”

• Take measurements of mutants’ physical and chemical parameters– e.g., plant height, leaf

size, root density, and nutrient utilization

• Different developmental times: compare to wild type

2002 Paradigm Genetics, Inc. All rights reserved. Used with permission.

Finding random mutations in your gene of interest (or every gene in

the genome)

• Random insertion of transposons

• Random point mutations/indels

Screening an insertion library

• PCR used to find insertion• One primer

complementary to insert• Other primer

complementary to gene• If get an amplification

product then you have insertion

• Sequence product for exact location

gene Z

insert

PCR primers

gene Z

insert

PCR amplification

+ –

amplification producton gel indicatespresence of insertnear gene

piggyBac

P element

Summary of P element Gene Disruption Project

TILLING

• Method for finding mutations produced by chemical mutagens in specific genes

• Chemical mutagenesis– Usually produces point mutations– Very high mutagenic efficiency– Generally gives more subtle phenotypes than

insertions• e.g., hypomorphs, temperature sensitive mutants

TILLING in Arabidopsis I

• EMS used to mutagenize Arabidopsis

• Grow individual mutagenized lines

• Make primers flanking gene of interest

• Amplify using PCRWT

mutant

gene Z

gene Z WT

mutant

PCR amplificationfrom wild typeand mutant

EMSmutagenizeseed

TILLING in Arabidopsis II• Denature DNA from pools

of mutant lines• Allow to hybridize to wild-

type DNA• Detect mismatches in

hybridized DNA– Denaturing HPLC– Cel I enzyme cuts at

mismatches

• Sequence to identify site of mutation

ATGCGGACTG|||||| |||TACGCCGGAC

ATGCGG CTG|||||| |||TACGCC GAC

Cel 1+

Arabidopsis TILLING Project

Summary I

• Forward genetics– Mutation to gene function– Genetic screens– Cloning genes identified in screens

• Genomics approaches to forward genetics– High-throughput genetic screens– Insertional mutagenesis– Activation tagging– Enhancer trapping and gene trapping