This document discusses strategies for genome-wide mutagenesis. It describes three main strategies: transposon insertion, gene disruption through allelic exchange, and expression inhibition using antisense RNA. Transposon insertion involves using transposable elements to randomly insert into genomes. Gene disruption uses targeted homologous recombination to replace genes. Antisense RNA inhibits gene expression by binding to target mRNA. The document also discusses various methods for detecting mutations, such as single-strand conformation polymorphism and allele-specific oligonucleotide hybridization.

1. 20 November 2014 1

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

3. CONTENTS
MUTATION
TYPES OF MUTATION
CAUSES OF MUTATIONS
PRODUCING MUTATIONS
STRATEGIES FOR GENOME-WIDE MUTAGENESIS
MUTATION DETECTION METHODS
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4. 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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5. 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 homozygous- usually
a loss of function.
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6. • 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- what’s lethal???
• Conditional: ts and nonsense mutations that are
suppressible
• Siamese cats have a ts mutation in a pigment-producing
gene; thus black paws, white body
(cooler paws, warmer body), with the black color
increasing in winter.
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7.  Point mutations
– silent mutation
– missense mutation
– nonsense mutation
– splicing mutation
 Rearrangements
– frameshift mutation
– codon deletion
– large deletion and
insertion
– deletions and
duplications
– trinucleotide
expansion
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8. 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, <106/gene/
generation 20 November 2014 8

9. Causes of Mutation
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10. 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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11. Tautomerization
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12. If the A switches back in
time, it produces a
mismatch, and proofreading
catches it- if not, it stays
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13. Deamination
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14. • D. Transposons/insertion sequences- jumping
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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15. II. Induced mutations:
• A. chemicals:1. Base analogs: increase in
tautomeric shifts, 5-Br uracil
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16. • 2. Alkylating agents: change H-bonding, labile
bonds with the sugar; induce SOS response,
which is mutagenic
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17. • 3. Intercalating agents: acridine dyes-increased
rate of frameshift mutations
Ethidium Bromide
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18. B. Radiation
• 1. UV light- Thymine dimers- again, repair can
be mutagenic
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19. • 2. gamma, X rays- DS and single-stranded
breaks; often deletions.
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20. Strategies For Genome Wide
Mutagenesis
Three major strategies for genome-wide
mutagenesis:
• transposon insertion,
• gene disruption by allelic exchange, and
• expression inhibition using antisense RNA
molecules.
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21. 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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22. • Contain two 9-
40bp copies of
terminally
inverted
nucleotide
repeats
• The inverted
repeats flank the
transposase gene.
Transposon
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23. Insertion sequence
• Have a central region carrying markers flanked by IS modules
• The IS arms are direct or inverted repeats
• Contains auxiliary genes unrelated to transposition
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24. • There are two major mechanisms for
transposition: conservative and replicative
transposition
Replicative transposition Conservative transposition
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25. 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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26. 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
expressed in subsequent
generations, resulting in
potential insertion instability
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27. B. In vitro Mutagenesis
• The in vitro approach is based on the ability of
purified transposases to catalyze strand-transfer
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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28. 20 November 2014 28

29. II.Targeted mutagenesis through allelic
exchange
1 Suicide Vector Systems for Allelic Exchange
Suicide plasmid’s 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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30. 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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31. B. Gene Replacement by Double-cross-over
Recombination: The Deletion–Substitution
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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32. 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:
• These results illustrate that in-frame, unmarked deletions are
among the most reliable types of mutations available for wild-type
E. coli.
The resulting PCR products were
placed in the E. coli chromosome by
using a gene replacement vector
Amplify target gene
(hdeA and yjbJ) by PCR
Two genes proved to be
nonessential
Replace chromosomal hdeA
with insertional alleles
Essential and nonessential
phenotypes were obtained
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33. 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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34. 2. Antisense Approach to Large-scale
Functional Genomic Studies
Genome-scale Antisense Silencing in S. aureus Using a Random
Antisense RNA Library
Figure : antisense m-RNA inhibition using the tc-inducible shuttle vector pyj335 20 November 2014 34

35. Gene Suppression in Candida albicans Using a
Combination of Antisense Silencing and Promoter
Interference
Figure : Integration of antisense library plasmids into C. albicans genome.
.
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36. 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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37. 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
– Allele specific oligonucleotide (ASO) probe
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38. Detecting Unknown Mutations
• Mutation screening
- SSCP
- Southern analysis
• Mutation confirming
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39. 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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40. Single-Strand Conformation
Polymorphism (SSCP)
Normal control Test (with mutation)
PCR products
Single strands
(conformers)
1. Amplify region to be scanned using PCR.
2. Denature and dilute
the PCR products.
3. Separate conformers by PAGE or CGE.
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41. Single-Strand Conformation
Polymorphism (SSCP)
4. Analyze results by comparison to reference normal control
(+).
PAGE CGE
+ mut +/mut +
mut
+/mut
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42. 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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43. 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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44. 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 m/+ +/+ m/m
+ probe m probe
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45. Allele-specific Oligomer Hybridization
(ASO)
• Chromogenic probe detection
– 1 – normal (+/+)
– 2 – heterozygous (m/+)
– m – heterozygous mutant control
– + – normal control
– N – negative control
1 2 m + N 1 2 m + N
+ probe m probe
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46. Melt Curve Analysis
• Based on sequence effect on Tm.
• Can be performed with or without probes.
• Requires double-strand DNA–specific dyes.
– Ethidium bromide
– SyBr Green
• Also performed with fluorescence resonance energy
transfer (FRET) probes.
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47. 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.
%SS
DS=SS
%DS
Fluorescence
Tm
50 Temperature (°C) 80
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48. Melt Curve Analysis
• Every sequence has a characteristic Tm.
• Melt curve Tm indicates which sequence is
present.
%SS
DS=SS
%DS
Heterozygous (m/+)
Homozygous
normal (+/+)
Homozygous
mutant (m/m)
50 80
Temperature (°C)
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49. Melt Curve Analysis
Detection instrument software may convert the melt
curve to a derivative of fluorescence (speed of drop
vs. temperature).
Temperature (°C)
Df/Dt
Normal
Heterozygous
mutant
Mutant Tm Normal Tm
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50. 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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51. 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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52. 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 single-base
differences in
DNA
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53. Sequence-Specific Primer PCR
(SSP-PCR)
PCR primer extension requires that the 3′ base
of the primer is complementary to the
template.
G
C
G
T
(Amplification)
(No amplification)
Normal template
Mutant template
Primer
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54. 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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55. 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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56. Allelic Discrimination
• Probe complementary to normal sequence
labeled with FAM fluorescent dye
• Probe complementary to normal sequence
labeled with VIC fluorescent dye
Normal Probe (FAM) Mutant Probe (VIC)
Normal
Mutant
Green signal
Red signal
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57. Allelic Discrimination
• Signals are detected and analyzed by the
instrument software.
• Multiple samples are analyzed simultaneously.
Normal allele (FAM)
Mutant allele
(VIC)
Mut
Het
NL
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58. Restriction Fragment Length
Polymorphism (RFLP)
• Restriction enzyme site recognition detects
presence of sequence changes.
e.g., G->A change creates EcoR1 site:
NL: … GTCA GGGTCC GTGC…
Mut: … GTCA GGATCC CTGC…
NL Mut Het
U C U C U C
Agarose
gel:
U – uncut
C – cut
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59. Heteroduplex Analysis with Single-
Strand Specific Nucleases
• Uses nucleases that cut single–stranded 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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60. Heteroduplex Analysis with Single-Strand
Specific Nucleases
Renature
Homoduplexes Heteroduplexes
Mutation
Mix, denature
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61. Heteroduplex Analysis with Single-Strand
Specific Nucleases
M NL Mutants
Heteroduplexes
cleaved by enzyme
Cleaved
fragments
indicate presence
of mutation
Homoduplexes
not cleaved by enzyme
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62. 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 complementary.
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63. Invader Technology
• Probes and enzyme are provided.
• 96-well plate format
A
T
mut probe
Cleavage
A
Complex formation
F Q
A
Cleavage F Detection
G
T
wt probe
(No cleavage)
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64. 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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65. CASE STUDY
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66. Target site duplication and footprints of Ac/Ds in rice.
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68. 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
reactivated through tissue culture. Use of progeny of
tissue culture-derived plants would make it possible to
screen revertants from mutant lines carrying inactive Ds.
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69. Videos
• 1
• 2
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70. REFERENCES
• http://www.lifetechnologies.com/castpcr
• https://www.youtube.com/watch?v=W6WA7l
M-sSQ
• www.ncbi.nlm.nih.gov/pubmed/9291975
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