Transcript Mutagenesis and Genetic Screens

Mutagenesis and Genetic
Screens
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”
Fig. 12-39
Classification of Mutant Type
Test for type of mutation recovered: haplo-insufficient null
Fig. 12-38
Test for type of mutation recovered: haplo-insufficient null
Fig. 12-38
Test for type of mutation recovered: haplo-insufficient leaky
Fig. 12-38
Test for type of mutation recovered: gain-of-function mutation
Fig. 12-38
Test for type of mutation recovered: neomorphic mutation
Fig. 12-38
General pathway for
mutational dissection
of a biological process
“Forward Genetics”
Fig. 12-39
From phenotype to gene
• Once an interesting
mutant is found and chromosome
characterized, we
want to find the gene
in which the mutant
occurred
• Positional cloning
– First use genetic
mapping
– Then use
chromosome walking
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 Para digm 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: 2002 Para digm Genetics, Inc. All rights reserved. Used with permission.
compare to wild type
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
PCR primers
insert
gene Z
PCR amplification
insert
gene Z
+
–
amplification product
on gel indicates
presence of insert
near gene
P element
piggyBac
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 PCR
EMS
mutagenize
seed
gene Z
WT
gene Z
mutant
PCR amplification
from wild type
and mutant
WT
mutant
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
||||||
TACGCC
CTG
|||
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