transposon mediated mutagenesis

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SCREENING AND GENERATION OF MUTANTS

PRESENTED BY:

SAURABH PANDEY

PALB-3252

CONTENTS

MUTATION

TYPES OF MUTATION

CAUSES OF MUTATIONS

PRODUCING MUTATIONS

STRATEGIES FOR GENOME-WIDE MUTAGENESIS

MUTATION DETECTION METHODS

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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 inducedspontaneous 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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• 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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Point mutations

– silent mutation

– missense mutation

– nonsense mutation

– splicing mutation

Rearrangements

– frameshift mutation

– codon deletion

– large deletion and insertion

– deletions and duplications

– trinucleotideexpansion

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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, <106/gene/ generation20 November 2014 8

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 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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II. Induced mutations:

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

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• 2. Alkylating agents: change H-bonding, 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

Ethidium Bromide20 November 2014 17

B. Radiation

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

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• 2. gamma, X rays- DS and single-stranded breaks; often deletions.

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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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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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• Contain two 9-

40bp copies of

terminally

inverted

nucleotide

repeats

• The inverted

repeats flank the

transposase gene.

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Transposon

• Have a central region carrying markers flanked by IS modules

• The IS arms are direct or inverted repeats

• Contains auxiliary genes unrelated to transposition20 November 2014 23

Insertion sequence

• There are two major mechanisms for transposition: conservative and replicative transposition

Conservative transpositionReplicative 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 MutagenesisAdvantage : 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 20 November 2014 26

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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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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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 Double-cross-over Recombination: The Deletion–Substitution

Method

Several other variations of the deletion–replacementmethod 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:

• 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 hdeAwith insertional alleles

Essential and nonessential phenotypes were obtained

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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 : antisense m-RNA inhibition using the tc-inducible shuttle vector pyj335 20 November 2014 34

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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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

– 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)

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.

Normal control Test (with mutation)

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+ mut +/mut +

mut

+/mut

4. Analyze results by comparison to reference normal control (+).

PAGE CGE

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5. Detect PAGE bands by silver staining.

T1 T2 NC

T1: test sample without mutationT2: test sample with mutationNC: 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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• 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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• Chromogenic probe detection

– 1 – normal (+/+)

– 2 – heterozygous (m/+)

– m – heterozygous mutant control

– + – normal control

– N – negative control

+ probe m probe

1 2 m + N 1 2 m + N

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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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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

Temperature (°C)50 80

Fluorescence

Tm

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Melt Curve Analysis

• Every sequence has a characteristic Tm.

• Melt curve Tm indicates which sequence is present.

%SS

DS=SS

%DS

50 80

Homozygous

normal (+/+)Homozygous mutant (m/m)

Heterozygous (m/+)

Temperature (°C)

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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

Mutant Tm Normal Tm

Heterozygous mutant

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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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Method Substrate Detection

Macroarray Nitrocellulose

Radioactive,

chemiluminescent,

chromogenic

MicroarrayGlass, nitrocellulose

on glassFluorescent

High-density

oligonucleotide

arrays

Glass Fluorescent

Microelectronic

arraysElectrode grid Fluorescent

Array Technologies

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Method Array Application

Comparative

genomic hybridization

(CGH)

Microarray,

macroarray

Detection of genomic

amplifications and

deletions

Expression arrayMicroarray,

macroarray

Detection of relative

changes in gene

expression

SNP detection,

mutation analysis,

sequencing

High density

oligonucleotide array

Detection of single-

base differences in

DNA

Microarray Technologies

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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.

G

C

G

T

(Amplification)

(No amplification)

Normal template

Mutant template

Primer

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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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• Probe complementary to normal sequence labeled with FAM fluorescent dye

• Probe complementary to normal sequence labeled with VIC fluorescent dye


Normal

Mutant

Normal Probe (FAM) Mutant Probe (VIC)

Green signal

Red signal

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• Signals are detected and analyzed by the instrument software.

• Multiple samples are analyzed simultaneously.

Normal allele (FAM)

Mu

tan

t al

lele

(VIC

)

Mut

NL

Het

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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 Het

U C U C U C

NL: … GTCA GGGTCC GTGC…Mut: … GTCA GGATCC CTGC…

Agarose gel:U – uncutC – cut

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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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Heteroduplex Analysis with Single-StrandSpecific Nucleases

Renature

HeteroduplexesHomoduplexes

MutationMix, denature

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Heteroduplex Analysis with Single-StrandSpecific Nucleases

M NL Mutants

Cleaved fragmentsindicate presenceof mutation

Heteroduplexescleaved by enzyme

Homoduplexesnot cleaved by enzyme

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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 complementary.

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Invader Technology

• Probes and enzyme are provided.

• 96-well plate format

A

T

mut probe

A

A

Cleavage

F Q

Complex formation

DetectionFCleavage

G

T

wt probe

(No cleavage)

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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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• 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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RESULT

Videos

• 1

• 2

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REFERENCES

• http://www.lifetechnologies.com/castpcr

• https://www.youtube.com/watch?v=W6WA7lM-sSQ

• www.ncbi.nlm.nih.gov/pubmed/9291975

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http://www.lifetechnologies.com/castpcr

https://www.youtube.com/watch?v=W6WA7lM-sSQ

http://www.ncbi.nlm.nih.gov/pubmed/9291975

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