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SCREENING AND
GENERATION OF MUTANTS
PRESENTED BY:
SAURABH PANDEY
PALB-3252

CONTENTS

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MUTATION
Sudden heritable changes in the
genetic material are called mutations.
A gene mutation is a change in the
nucleotide sequence that composes a
gene. This is a change or variation
from the most common or wildtype
sequence.

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TYPES OF MUTATION
Spontaneous or induced
spontaneous by the natural forces and
induced due to mutagens.
Somatic or Germ line
Dominant or recessive
dominant mutation in a non-sex
chromosome; expresses when heterozygous;
overproduction or gain of function.
Recessive: expressed when homozygoususually a loss of function.
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 X-linked recessive- recessive in females, dominant

in males because of only one X chromosome.
Other categories:
Morphological- six fingers, achondroplasia
(dwarfism)
Nutritional: prototroph vs auxotroph (mostly
bacteria and fungi)
Lethal- whats lethal???
Conditional: ts and nonsense mutations that are
suppressible
Siamese cats have a ts mutation in a pigmentproducing gene; thus black paws, white body
(cooler paws, warmer body), with the black color
increasing in winter.

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Point mutations

silent mutation
missense mutation
nonsense mutation
splicing mutation

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Rearrangements
frameshift
mutation
codon deletion
large deletion
and insertion
deletions and
duplications
trinucleotide
expansion
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What causes mutations?

1. Spontaneous- wide variety of mutations types
substitutions, deletions, frameshifts, insertions
2. DNA replications errors- not repaired
3. Recombination > rearrangements-> deletions
and insertions (duplications)
4. DNA damage radiation, metabolisms, free
radicals
5.
Transposable elements insertions, usually rare,8
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<1066/gene/ generation

Causes of Mutation

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Producing mutations- spontaneous

and induced
I. Spontaneous-infrequent. one natural
cause:
A. tautomerization B. Deamination: cytosine-->uracil;
adenine--->hypoxanthine, acts like a
G.
C. Environmental effects: sunlight,
cosmic rays.
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Tautomerization

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If the A switches back

in time, it produces a
mismatch, and
proofreading catches
it- if not, it stays

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Deamination

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 D. Transposons/insertion sequencesjumping genes

E. Replication/repair defects- human
diseases with trinucleotide repeats.
There are a number of human
genetic conditions- Huntington and
fragile X syndrome are the best
known- which are caused by an
excess in trinucleotide repeats.
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II. Induced mutations:

A. chemicals:1. Base analogs:
increase in tautomeric shifts, 5-Br
uracil

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 2. Alkylating agents: change Hbonding, labile bonds with the sugar;

induce SOS response, which is
mutagenic

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 3. Intercalating agents: acridine

dyes- increased rate of frameshift
mutations

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Ethidium Bromide

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B. Radiation
1. UV light- Thymine dimers- again,
repair can be mutagenic

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 2. gamma, X rays- DS and singlestranded breaks; often deletions.

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Strategies For Genome Wide

Mutagenesis
Three major strategies for genomewide mutagenesis:
transposon insertion,
gene disruption by allelic exchange,
and
expression inhibition using antisense
RNA molecules.

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I.Transposon
Mutagenesis
1.Overview of transposition in
bacteria
classification of transposable
elements
There are two major groups in
bacteria :Insertion sequence (IS) and
transposons (Tn)
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Transposon
Contain two
9-40bp copies
of terminally
inverted
nucleotide
repeats
The inverted
repeats flank
the
transposase
gene.
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Insertion
sequence

Have a central region carrying markers flanked by

IS modules
The IS arms are direct or inverted repeats

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2/4/15Contains auxiliary genes unrelated to transposition

 There are two major mechanisms for

transposition: conservative and
replicative transposition
Replicative transposition

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Conservative transposition

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Transposon Delivery System

Include suicide phages and plasmids
that are unable to replicate within
the host strain, but possess
mobilization ability and a broad host
range of transfer.
The choice of a delivery vehicle
largely depends on the properties of
the recipient strain and on the
transposition target.
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Transposons as Tools for Mutagenesis

A. In vivo Mutagenesis
Advantage : The target
organism does not have
to be naturally
competent
Disadvantage: The
transposon must be
introduced into the host
on a suicide vector, the
transposase must be
expressed in the target
host, and the
transposase usually is
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expressed
in

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B. In vitro Mutagenesis
The in vitro approach is based on the ability of
purified transposases to catalyze strandtransfer reactions between linear DNA
molecules in a cell-free environment .
Advantages: it have the ability to reach high
saturation levels of mutagenesis, which allows
one to conduct analyses of the target locus on
either large or small scales.
Disadvantage: it have the prerequisite for
preliminary information on the target
sequence.
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II.Targeted
mutagenesis through allelic
exchange
1

Suicide Vector Systems for Allelic

Exchange
Suicide plasmids properties:

It is conditional for replication to

allow selection for integration into
the chromosome
It carry a selectable marker
It should be transferable to a wide
variety of organisms
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2 Strategies Commonly Utilized for

Targeted Mutagenesis by Allelic Exchange

A. Integration of Conditional
Replicons by Single-cross-over
Recombination: The Insertion
Duplication Method

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B. Gene Replacement by Doublecross-over Recombination: The

DeletionSubstitution Method

Several other variations of the deletion

replacement method have been developed
using plasmids of the IncP incompatibility groups
transform linear DNA substrates into the organism of
interest.

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Application of Allele Exchange Approach in

Functional Genomic Studies for Sequenced
Microorganisms

Characterization of Unknown Genes in E. coli

Using In-frame Precise Deletions
PCR-based in-frame deletion system:
The resulting PCR products
were placed in the E. coli
chromosome by using a gene
replacement vector
Two genes proved to be
nonessential

Amplify target gene

(hdeA and yjbJ) by
PCR

Replace chromosomal
hdeA with insertional
alleles

Essential and nonessential

phenotypes were obtained

These results illustrate that in-frame, unmarked

deletions are among the most reliable types of
mutations available for wild-type E. coli.
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III.Gene silencing using

antisense mRNA molecules
Antisense RNA regulation in vivo
(1) translation blockage by antisense hybridization
to target mRNAs
(2) translation initiation inhibition by occlusion of the
ribosome binding site
(3) premature termination of mRNA transcription due
to antisense binding to the genomic DNA template
(4) stimulation of rapid mRNA degradation by
duplex-specific Rnases ,and
(5) reduction of enzymatic activity by antisense
binding to the target protein
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2. Antisense Approach to Large-scale Functional Genomic

Studies

Genome-scale Antisense Silencing in S.

aureus Using a Random Antisense RNA
Library

Figure
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vector

: antisense m-RNA inhibition using the tc-inducible shuttle

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pyj335

Gene Suppression in Candida

albicans Using a Combination of
Antisense Silencing and Promoter
Interference

Figure : Integration of antisense library plasmids into C.

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albicans genome.

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Types of Mutation Detection

Methods
Hybridization-based
SSCP, ASO, melt curves, array
technology
Sequencing (polymerization)-based
Sequence-specific PCR, allelic
discrimination
Cleavage-based
RFLP, nuclease cleavage, invader
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Detecting Known Mutations

Insertion or deletion
large fragments by Southern
small fragments by PCR
Point mutation
Restriction site altered by mutation
RFLP or PCR/restriction enzyme
digestion

No restriction site altered by

mutation
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Allele specific oligonucleotide (ASO)

probe

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Detecting Unknown
Mutations
Mutation screening
- SSCP
- Southern analysis
Mutation confirming

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Single-Strand Conformation
Polymorphism
Scans several-hundred base pairs.
Based on intra-strand folding.
Single strands will fold based on
sequence.
One base difference will affect folding.

Folded single strands (conformers)

can be resolved by size and shape.
Strict temperature requirements.
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Single-Strand Conformation
Polymorphism (SSCP)
1. Amplify region to be scanned using PCR.
Normal control
mutation)

2. Denature and
dilute the PCR
products.

Test (with
PCR products

Single strands
(conformers)

3. Separate conformers by PAGE or CGE.

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Single-Strand Conformation
Polymorphism (SSCP)
4. Analyze results by comparison to reference
normal control (+).
+

PAGE
CGE

mut

+/mut

mut
+/mut

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Single-Strand Conformation
Polymorphism (SSCP)
5. Detect PAGE bands by silver staining.
T1 T2 NC

T1: test sample without mutation

T2: test sample with mutation
NC: normal control

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Allele-specific Oligomer
Hybridization (ASO)
Dot blot method
Relies on binding effects of
nucleotide mismatches.
Specimen in solution is spotted on
nitrocellulose.
Labeled oligonucleotide probe is
hybridized to immobilized specimen.
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Allele-specific Oligomer
Hybridization (ASO)
Three specimens spotted on duplicate
membranes.
One membrane exposed to probe
complementary to the normal sequence (+
probe).
One membrane exposed to probe
complementary to the mutant sequence (m
probe).
m/+ +/+ m/m
+ probe
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m/+ +/+ m/m

m probe
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Allele-specific Oligomer
Hybridization (ASO)
Chromogenic probe detection

1 normal (+/+)
2 heterozygous (m/+)
m heterozygous mutant control
+ normal control
N negative control
1

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m +

+ probe

m probe

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Melt Curve Analysis

Based on sequence effect on Tm.
Can be performed with or without probes.
Requires double-strand DNAspecific
dyes.
Ethidium bromide
SyBr Green

Also performed with fluorescence

resonance energy transfer (FRET )
probes.
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Melt Curve Analysis

Double-stranded DNA specific dye (SyBr
Green) will fluoresce when bound to DNA.
Denaturation of DNA to single strands will
result in loss of fluorescence.
Fluorescence
%SS

DS=SS

Tm

%DS
50
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Temperature (C)

80
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Melt Curve Analysis

Every sequence has a characteristic
Tm.
Melt curve Tm indicates which
Heterozygous (m/+)
sequence is present.
%SS

DS=SS

%DS

Homozygo
us
mutant
(m/m)
50

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Temperature (C)

Homozygous
normal (+/+)

80
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Melt Curve Analysis

Df/Dt

Detection instrument software may

convert the melt curve to a derivative of
fluorescence (speed of drop vs.
Normal
temperature).

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Heterozygous
mutant

Temperature (C)
Mutant Tm
Normal
Tm

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Array Technology
Reverse dot blot methods.
Used to investigate multiple
genomic sites simultaneously.
Unlabeled probes are bound to
substrate.
Specimen DNA is labeled and
hybridized to immobilized probes.
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Array Technologies
Method

Substrate

Detection

Macroarray

Nitrocellulose

Radioactive,
chemiluminescent,
chromogenic

Microarray

Glass, nitrocellulose
on glass

Fluorescent

High-density
oligonucleotide
arrays

Glass

Fluorescent

Microelectronic
arrays

Electrode grid

Fluorescent

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Microarray Technologies
Method

Array

Application

Comparative
genomic
hybridization (CGH)

Microarray,
macroarray

Detection of genomic
amplifications and
deletions

Expression array

Microarray,
macroarray

Detection of relative
changes in gene
expression

SNP detection,
mutation analysis,
sequencing

High density
oligonucleotide array

Detection of singlebase differences in

DNA

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Sequence-Specific Primer PCR

(SSP-PCR)
PCR primer extension requires that
the 3 base of the primer is
complementary to the template.
Primer
G
C

Normal template

G
Mutant template
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(Amplification)

(No amplification)
T
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Sequence-Specific Primer PCR

(SSP-PCR)
Primer design is used to detect
mutations in DNA.
Generation of PCR product indicates
the presence of mutation or
polymorphism in the template.

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Allelic Discrimination
Uses fluorescently labeled probes.
Similar to Taqman technology.
Generates color signal for mutant
or normal sequence.
Performed on real-time PCR
instruments.

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Allelic Discrimination
Probe complementary to normal
sequence labeled with FAM
fluorescent dye
Probe complementary to normal
sequence labeled with VIC
fluorescent
Normal Probe (FAM) dye
Mutant Probe (VIC)
Normal

Green signal

Mutant

Red signal

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Allelic Discrimination

Mutant allele
(VIC)

Signals are detected and analyzed by

the instrument software.
Multiple samples are analyzed
simultaneously.
Het
Mut

NL
Normal allele (FAM)

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Restriction Fragment Length

Polymorphism (RFLP)
Restriction enzyme site recognition
detects presence of sequence changes.
e.g., G->A change creates EcoR1 site:
NL:
Mut:

GTCA GGGTCC
GTCA GGATCC

NL
U

Mut
C

GTGC
CTGC
Het
U
C

Agarose
gel:
U uncut
C cut
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Heteroduplex Analysis with

Single-Strand Specific Nucleases
Uses nucleases that cut singlestranded
bubbles in heteroduplexes.
Region of interest is amplified by PCR.
PCR product is denatured and renatured
with or without added normal PCR
product.
Renatured duplexes are digested with
nuclease; e.g., S1 nuclease
Products are observed by gel
electrophoresis.
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Heteroduplex Analysis with

Single-Strand Specific
Nucleases
Mutation

Mix, denature

Renature

Homoduplexes
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Heteroduplexes
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Heteroduplex Analysis with

Single-Strand Specific Nucleases
Homoduplexes
not cleaved by enzyme
M

NL

Heteroduplexes
cleaved by enzyme

Mutants
Cleaved
fragments
indicate presence
of mutation

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Invader Technology
Mutation detection with proprietary
Cleavase enzyme.
Sample is mixed with probes and
enzyme.
Enzyme cleavage of probe-test
sample hybrid will yield fluorescent
signal.
Signal will only occur if probe and
test sample sequence are

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Invader Technology
Probes and enzyme are provided.
96-well plate format
A

mutprobe
G

T
Cleavage
A

Complex formation
F

wt probe

(No cleavage)

Q
A

Cleavage
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Detection
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Summary
Mutations and polymorphisms are
changes in the DNA sequence.
DNA sequence changes have
varying effects on the phenotype.
Molecular detection of mutations
include hybridization-, sequence-, or
cleavage- based methods.
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CASE STUDY

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Target site duplication and footprints of Ac/Ds in

rice.

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RESULT
The maize Ac/Ds system is an effective
mutagen for rice.
However, no linkage of Ds elements with the
mutant phenotypes indicates that integration
and excision of Ds in F1 plants might be too
frequent to identify a linked Ds using the
segregating populations originated from F2
lines.
Southern blot analysis using Ds as a probe
revealed that inactivation of Ds transposition
was often observed in F3 and F4 generations.
To overcome this potential obstacle, we
demonstrated
that these inactive Ds can be 68
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reactivated through tissue culture. Use of

Videos
1

TaqMan and castPCR for Somatic Mutation Detection in Cancer Genes.mp4

2
Screening Mutations.mp4

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REFERENCES
http://www.lifetechnologies.com/cast
pcr
https://www.youtube.com/watch?v=
W6WA7lM-sSQ
www.ncbi.nlm.nih.gov/pubmed/9291
975

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