Skip to main content

Site-Directed Mutagenesis (SDM) – Detailed Notes


Site-Directed Mutagenesis (SDM) – Detailed Notes


1. Definition

Site-Directed Mutagenesis (SDM) is a molecular biology technique used to introduce specific, predetermined changes (mutations) at a precise location in a DNA sequence.
It allows controlled alteration of genes, enabling scientists to study gene function, protein structure, and enzyme activity.
Mutations that can be introduced:
Substitution: Change of a single nucleotide
Insertion: Addition of nucleotides
Deletion: Removal of nucleotides

2. Principle

The principle of SDM involves using a DNA template and synthetic oligonucleotide primers that carry the desired mutation.
These primers anneal to the complementary DNA and are extended using DNA polymerase, generating a mutated copy.
The original template is usually digested or not propagated, leaving only the mutated DNA.


Key Concept:


By introducing a mutation in a primer that binds the DNA template, the polymerase incorporates this change during DNA replication, producing a mutated gene.


3. Types of Mutations Introduced
Point Mutation (Base Substitution):

Changes a single nucleotide.
Example: Codon AAA → AGA changes lysine to arginine.

Deletion Mutation:
Removes one or more nucleotides from the gene.
Example: Removing a codon to delete a specific amino acid.


Insertion Mutation:

Adds one or more nucleotides.
Example: Adding a codon to insert an amino acid.

Multiple Mutations:

Introducing more than one mutation at different sites in the gene.

4. Methods of Site-Directed Mutagenesis

A. Primer Extension Method
One of the earliest methods, also known as the Kunkel method.
Uses single-stranded DNA template and mutagenic primers.
Steps:
Denature template DNA.
Anneal primer with mutation.
Extend primer using DNA polymerase.
Ligate and transform into host cells.

B. PCR-Based Methods


Overlap Extension PCR (OE-PCR):
Two DNA fragments with overlapping ends containing the mutation are amplified.
Fragments anneal and are extended to generate full-length mutated DNA.
Can introduce substitutions, insertions, or deletions.

Megaprimer PCR Method:

A PCR product containing the mutation (megaprimer) is used to amplify the entire gene.
Efficient for single mutations.

QuikChange Method:

Widely used commercially.
Uses mutagenic primers and high-fidelity DNA polymerase.
Methylated template DNA is digested using DpnI, leaving only mutated plasmid.


C. Cassette Mutagenesis


A synthetic DNA fragment (cassette) containing the mutation is inserted into the target gene using restriction enzymes and ligation.
Useful for large insertions, deletions, or replacements.

5. Requirements for SDM

Template DNA: Plasmid containing the target gene.
Mutagenic primers:
Contain the desired mutation.
Typically 25–45 nucleotides long with stable GC-rich 3′ ends.

DNA Polymerase: High-fidelity enzyme to minimize unwanted mutations.

Host cells: Usually E. coli for propagation of mutated plasmids.
Selection method: Antibiotic resistance or screening assays to identify mutated clones.


6. Applications of SDM

Protein Engineering:
Alter enzyme activity, substrate specificity, or thermostability.
Functional Genomics:
Determine the role of specific amino acids in protein function.
Gene Regulation Studies:
Alter promoter or enhancer sequences to study gene expression.
Drug Development:
Introduce mutations in target genes to study drug resistance mechanisms.
Vaccine Development:
Generate attenuated viruses with specific mutations.
Metabolic Engineering:
Modify metabolic enzymes to enhance industrial or agricultural productivity.
7. Advantages of SDM

Precise and specific changes at predetermined sites.
Can introduce point mutations, insertions, deletions.
Minimal off-target effects.
Allows structure-function analysis of proteins.
Useful in drug and vaccine development.


8. Limitations of SDM


Requires knowledge of the DNA sequence.
Primer design and polymerase selection affect efficiency.
Introducing multiple mutations may require complex strategies.
Some mutations may affect plasmid stability or cell viability.
Screening for mutated clones can be labor-intensive.


9. Examples


Ξ²-galactosidase mutation: Study active site amino acids.
HIV reverse transcriptase mutation: Understand drug resistance.
Engineering Taq DNA polymerase: Increase thermostability.
Green Fluorescent Protein (GFP) mutation: Create fluorescent variants.


Applications

Protein engineering, functional genomics, drug/vaccine development, metabolic engineering


Advantages

Precise, versatile, minimal off-target effects, structure-function analysis
Limitations
Requires sequence info, multiple mutations challenging, screening needed




Site-Directed Mutagenesis (SDM) – 50 MCQs


What is the main purpose of site-directed mutagenesis?


a) Random mutation of DNA
b) Introduce a specific mutation at a defined site ✅
c) Amplify RNA
d) Clone proteins

Which type of mutation can be introduced by SDM?
a) Substitution ✅
b) Deletion ✅
c) Insertion ✅
d) All of the above ✅


SDM is primarily used to study:
a) Protein structure-function relationships ✅
b) Chromosome number
c) RNA splicing
d) Membrane transport
Which type of primer is used in SDM?
a) Random primer
b) Mutagenic primer ✅
c) RNA primer
d) Ribosomal primer
In QuikChange SDM, which enzyme digests the template DNA?
a) EcoRI
b) DpnI ✅
c) DNA ligase
d) Taq polymerase
Which polymerase is preferred for SDM?
a) Low-fidelity polymerase
b) High-fidelity DNA polymerase ✅
c) RNA polymerase
d) Reverse transcriptase
The Kunkel method uses:
a) Double-stranded DNA
b) Single-stranded DNA template ✅
c) RNA template
d) Protein template
Overlap Extension PCR (OE-PCR) is used to introduce:
a) Point mutations ✅
b) Insertions ✅
c) Deletions ✅
d) All of the above ✅
In cassette mutagenesis, the mutation is introduced using:
a) PCR primers
b) Synthetic DNA fragment ✅
c) Random mutagen
d) RNA oligonucleotides
Which of the following is an example of SDM application?
a) Studying enzyme active site ✅
b) DNA fingerprinting
c) RNA interference
d) Protein crystallography
SDM allows mutation at:
a) Random sites
b) Predetermined sites ✅
c) Promoter regions only
d) Exons only
A point mutation involves:
a) Substitution of one nucleotide ✅
b) Addition of a codon
c) Deletion of a gene
d) Frame-shift of multiple nucleotides
Insertion mutation in SDM leads to:
a) Removal of nucleotides
b) Addition of nucleotides ✅
c) Change of a single base
d) Protein degradation
Deletion mutation in SDM leads to:
a) Addition of nucleotides
b) Removal of nucleotides ✅
c) RNA synthesis
d) DNA replication
Which SDM method uses high-fidelity PCR with mutagenic primers and DpnI digestion?
a) Kunkel method
b) QuikChange ✅
c) OE-PCR
d) Cassette mutagenesis
Mutagenic primers usually have:
a) 10–15 nucleotides
b) 25–45 nucleotides ✅
c) 50–70 nucleotides
d) Only 5 nucleotides
Which of the following can SDM NOT introduce?
a) Substitution
b) Deletion
c) Duplication ✅
d) Insertion
SDM is widely used in:
a) Protein engineering ✅
b) Protein folding studies
c) Chromosome staining
d) RNA extraction
Which method allows multiple mutations at once?
a) Kunkel method
b) OE-PCR ✅
c) QuikChange
d) Primer walking
Cassette mutagenesis is most useful for:
a) Small point mutations
b) Large insertions or replacements ✅
c) Single nucleotide deletions
d) Random mutagenesis
The success of SDM depends largely on:
a) DNA template quality ✅
b) Gel electrophoresis
c) RNA primers
d) Protein folding
High GC content in primer 3′ end ensures:
a) Primer degradation
b) Stable annealing ✅
c) Random mutation
d) Template digestion
DpnI enzyme cuts:
a) Unmethylated DNA
b) Methylated template DNA ✅
c) RNA
d) Protein
SDM can be used to alter:
a) Enzyme activity ✅
b) Ribosomal RNA
c) Membrane fluidity
d) Cell wall composition
PCR-based SDM is preferred over Kunkel because:
a) Requires single-stranded DNA
b) Faster and high-throughput ✅
c) Does not require primers
d) Works without polymerase
In OE-PCR, the mutated region is introduced using:
a) Overlapping primers ✅
b) Random primers
c) Restriction enzymes only
d) RNA polymerase
Site-directed mutagenesis helps in understanding:
a) Gene regulation ✅
b) Cell division
c) Mitochondrial metabolism
d) Protein folding only
Megaprimer PCR method uses:
a) A PCR product as a primer ✅
b) RNA as a primer
c) Protein as a primer
d) Random oligonucleotides
SDM is different from random mutagenesis because:
a) Mutations occur randomly
b) Mutations are predefined ✅
c) Only insertions occur
d) Only deletions occur
Which is a key requirement for SDM?
a) Knowledge of DNA sequence ✅
b) Knowledge of RNA sequence
c) Protein structure
d) Chromosome number
QuikChange mutagenesis is commercially available as:
a) PCR kit ✅
b) Restriction enzyme kit
c) Sequencing kit
d) Protein purification kit
SDM can be applied in drug resistance studies by:
a) Mutating target genes ✅
b) Deleting plasmids
c) RNA interference
d) Gel electrophoresis
A major advantage of SDM is:
a) Random mutation
b) Specificity and precision ✅
c) Requires no primers
d) Works only in eukaryotes
SDM can also be used in:
a) Vaccine development ✅
b) RNA transcription
c) Protein sequencing
d) Chromosome mapping
Screening of mutated clones is required because:
a) Not all plasmids get mutated ✅
b) All plasmids are mutated
c) Template DNA is always degraded
d) Polymerase does not work
Which polymerase is used to reduce unwanted mutations?
a) Taq polymerase
b) High-fidelity DNA polymerase ✅
c) Reverse transcriptase
d) RNA polymerase
Point mutation changes:
a) One nucleotide ✅
b) Codon triplet
c) Entire gene
d) Protein only
Large deletions or insertions are easier with:
a) Kunkel method
b) Cassette mutagenesis ✅
c) QuikChange
d) Random mutagenesis
SDM can be used to study:
a) Structure-function of enzymes ✅
b) RNA splicing
c) DNA replication rate
d) Membrane lipid composition
Screening of SDM clones may involve:
a) Antibiotic resistance ✅
b) RNA extraction
c) Protein crystallization
d) DNA fingerprinting
OE-PCR uses:
a) Single primer
b) Two overlapping primers ✅
c) Restriction enzymes only
d) RNA primer
QuikChange relies on:
a) Single-stranded DNA
b) Double-stranded DNA template ✅
c) RNA template
d) Random oligonucleotides
A limitation of SDM is:
a) Requires DNA sequence knowledge ✅
b) Can only introduce deletions
c) Only works in RNA
d) Cannot introduce point mutations
SDM can modify which types of genes?
a) Bacterial ✅
b) Viral ✅
c) Eukaryotic ✅
d) All of the above ✅
Mutagenic primers must:
a) Contain the desired mutation ✅
b) Be entirely complementary to template
c) Not bind template
d) Only bind RNA
High GC content in primer 3′ end prevents:
a) Primer slippage ✅
b) DNA replication
c) Protein synthesis
d) RNA binding
SDM is important in enzyme engineering to:
a) Study active site ✅
b) Measure molecular weight
c) Sequence RNA
d) Purify protein
Multiple mutations require:
a) Single primer
b) Complex PCR strategies ✅
c) No primers
d) Only ligase
Screening of SDM clones can involve:
a) DNA sequencing ✅
b) Northern blot
c) Protein ELISA
d) RNA extraction
SDM helps to understand:
a) Gene function ✅
b) Chromosome number
c) Membrane potential
d) Cell wall thickness

Comments

Post a Comment

Popular Posts

❥ Southern Blotting Notes

Southern Blotting  ❥ π“†ž❥ π“†ž❥ π“†ž❥ π“†ž❥ π“†ž❥ π“†ž❥ π“†ž❥ π“†ž❥ π“†ž❥  Introduction Southern blotting is a molecular biology technique used for the detection of specific DNA sequences in a complex mixture of DNA. It was developed by Edwin M. Southern in 1975. The method involves restriction digestion of DNA, separation by gel electrophoresis, transfer (blotting) onto a membrane, and hybridization with a labeled DNA probe. Principle of Southern Blotting The technique is based on the principle of complementary base pairing. A single-stranded labeled DNA probe hybridizes specifically with its complementary DNA sequence immobilized on a membrane. Detection of the label confirms the presence and size of the target DNA fragment. Steps Involved in Southern Blotting. 1. Isolation of DNA Genomic DNA is extracted from cells or tissues. DNA must be pure and intact to ensure accurate results. 2. Restriction Enzyme  Digestion DNA is digested using specific restriction endonucleases. Produces DNA f...
1 comment
Read more

AFLP--Amplified Fragment Length Polymorphism

AFLP is a PCR-based DNA fingerprinting technique combining restriction digestion and selective PCR amplification of genomic DNA fragments. Developed by Vos et al., 1995. AFLP detects DNA polymorphisms at the genomic level and is highly reproducible and sensitive. Used in genetic mapping, diversity studies, phylogenetics, and marker-assisted selection. Principle AFLP relies on restriction digestion of genomic DNA, followed by ligation of adaptors and PCR amplification of a subset of fragments. Polymorphism arises due to variations in restriction sites, fragment length, insertions, or deletions. Key idea: Restriction digestion → Adaptor ligation → Selective amplification → Gel separation → Detection of polymorphic bands Materials Required Genomic DNA Restriction enzymes (usually EcoRI and MseI) Adaptors complementary to restriction sites PCR reagents: Taq polymerase, dNTPs, buffer, Mg²⁺ Primers complementary to adaptors with selective nucleotides Thermal cycler Polyacrylamide or agarose ...
Post a Comment
Read more

GEL RETARDATION ANALYSIS

GEL RETARDATION ANALYSIS (EMSA – Electrophoretic Mobility Shift Assay) Introduction Gel retardation analysis, also known as Electrophoretic Mobility Shift Assay (EMSA), is a widely used in vitro technique for studying DNA–protein and RNA–protein interactions. The method is based on the observation that a DNA–protein complex migrates more slowly than free DNA during non-denaturing gel electrophoresis, resulting in a mobility shift or “retardation”. EMSA is extensively used to study transcription factor binding, regulatory DNA elements, and binding specificity. Definition Gel retardation analysis (EMSA) is a technique used to detect and analyze binding interactions between nucleic acids and proteins by observing the reduced electrophoretic mobility of nucleic acid–protein complexes compared to free nucleic acids. Principle A labeled DNA or RNA probe is incubated with a specific binding protein. When binding occurs, a nucleic acid–protein complex is formed. This complex has a larger size ...
Post a Comment
Read more

DNA-Mediated Gene Transfer – Detailed Notes

DNA-Mediated Gene Transfer – Detailed Notes 1. Definition DNA-mediated gene transfer refers to the direct introduction of exogenous DNA into a host cell’s genome or cytoplasm without using viral or bacterial vectors. It is a physical or chemical approach to achieve gene delivery. Also called direct gene transfer. 2 . Principle Foreign DNA is delivered into host cells through physical or chemical methods. DNA may integrate into the host genome (stable transformation) or remain episomal (transient expression). Expression depends on: DNA sequence and promoter Type of host cell Delivery efficiency 3. Types of DNA-Mediated Gene Transfer A. Physical Methods These methods use physical forces to introduce DNA into cells. Microinjection DNA is injected directly into the nucleus or cytoplasm using a glass micropipette. Used in: animal embryos, oocytes, plant protoplasts Advantages: Precise, can deliver large DNA fragments Limitations: Labor-intensive, requires specialized equipment, low throughp...
Post a Comment
Read more

Agrobacterium & CaMV-Mediated Gene Transfer –

Agrobacterium and CaMV-Mediated Gene Transfer – Detailed Notes 1. Introduction Gene transfer in plants is often achieved by exploiting natural genetic mechanisms of Agrobacterium tumefaciens and Cauliflower Mosaic Virus (CaMV). These systems allow stable introduction of foreign genes into plant genomes for transgenic plant development. 2. Agrobacterium-Mediated Gene Transfer 2.1 Definition Agrobacterium-mediated gene transfer uses the natural ability of Agrobacterium tumefaciens, a soil bacterium, to transfer a part of its DNA (T-DNA) into plant cells. T-DNA integrates into the plant nuclear genome, enabling stable transformation. 2.2 Mechanism Recognition and attachment Agrobacterium detects phenolic compounds secreted by wounded plant cells. These compounds activate virulence (vir) genes on the Ti (tumor-inducing) plasmid. Activation of vir genes VirA (sensor kinase) and VirG (response regulator) induce expression of other vir genes (VirB, VirC, VirD, VirE). T-DNA processing and tran...
Post a Comment
Read more

❥NORTHERN BLOTTING

NORTHERN BLOTTING – 30 MARK DETAILED NOTES  π“†ž❥ π“†ž❥ π“†ž❥ π“†ž❥ π“†ž❥ π“†ž ❥ π“†ž❥ π“†ž❥  Northern blotting is a molecular biology technique used to detect specific RNA molecules in a complex mixture. It provides information about gene expression, RNA size, and transcript abundance by hybridizing RNA with a labeled complementary DNA or RNA probe. πŸ“Œ Named by analogy to Southern blotting (DNA detection). 2. Principle The principle of Northern blotting is based on: Separation of RNA molecules by size using denaturing agarose gel electrophoresis Transfer (blotting) of separated RNA onto a nylon or nitrocellulose membrane Hybridization of membrane-bound RNA with a labeled complementary probe Detection of RNA–probe hybrids by autoradiography or chemiluminescence ✔ Only RNA sequences complementary to the probe will be detected. 3. Types of RNA Analyzed mRNA (most common) rRNA tRNA miRNA and siRNA (with modified protocols) 4. Requirements / Materials Total RNA or poly(A)+ RNA Denaturing agarose ...
Post a Comment
Read more

❥ preservation for germplasm conservation. Cryopreservation of vegetative propagated and recalcitrant seed species.

preservation for germplasm conservation. Cryopreservation of vegetative propagated and recalcitrant seed species.  ❥ π“†ž❥ π“†ž❥ π“†ž❥ π“†ž❥ π“†ž❥ π“†ž❥ π“†ž❥ π“†ž❥ π“†ž❥ 1. Introduction Germplasm conservation is the systematic preservation of genetic resources for present and future use. Many economically important plants are vegetatively propagated (banana, potato, sugarcane, cassava) or produce recalcitrant seeds (cocoa, rubber, coconut), which cannot be conserved by conventional seed storage. Cryopreservation offers a safe, long-term and genetically stable method for conserving such germplasm by storing living tissues at –196°C in liquid nitrogen (LN). 2. Need for Preservation of Germplasm Prevention of genetic erosion Conservation of elite, endangered and rare species Backup of field and in-vitro collections Support to crop improvement and breeding Preservation of pathogen-free planting material Conservation of plants with non-orthodox seeds 3. Limitations of Conventional Storage Methods Seed B...
Post a Comment
Read more

Protoplast culture covering isolation, fusion, somatic hybrid & cybrid production, preferential chromosome elimination, role in CMS, and genetic transformation.

  Protoplast culture covering isolation, fusion, somatic hybrid & cybrid production, preferential chromosome elimination, role in CMS, and genetic transformation. Protoplast Culture 1. Introduction A protoplast is a plant cell without a cell wall, surrounded only by the plasma membrane. Protoplast culture allows direct access to the plasma membrane and genome, making it a powerful tool for: Somatic hybridization Cybrid production Genetic transformation Cytoplasmic trait transfer (e.g., CMS) 2. Isolation of Protoplasts 2.1 Source of Protoplasts Young leaves (mesophyll cells) Callus tissue Cell suspension cultures Roots or hypocotyls Young, actively dividing tissues are preferred due to high viability. 2.2 Methods of Protoplast Isolation A. Mechanical Method Cell walls removed by cutting and plasmolysis Rarely used Causes low yield and high damage B. Enzymatic Method (Most Common) Cell wall digested using enzymes: Enzyme Function Cellulase Degrades cellulose Pectinase Degrades mi...
Post a Comment
Read more

Secondary Databases (PROSITE, PRINTS, BLOCKS)

Secondary Databases (PROSITE, PRINTS, BLOCKS  Secondary Databases Introduction Biological databases are broadly classified into primary and secondary databases. Primary databases store raw experimental data (e.g., nucleotide or protein sequences), whereas secondary databases contain derived information obtained by analyzing primary sequence data. Secondary databases are mainly used to: Identify protein families Detect conserved motifs, patterns, and domains Predict protein function Study structure–function relationships Examples of secondary databases include PROSITE, PRINTS, BLOCKS, Pfam, etc. 1. PROSITE Database Definition PROSITE is a secondary database that documents protein domains, families, and functional sites in the form of patterns and profiles. Developed by Swiss Institute of Bioinformatics (SIB) Maintained along with UniProt Principle PROSITE is based on the idea that functionally important regions of proteins are conserved during evolution. These conserved regions can ...
Post a Comment
Read more

Gene Transfer Technologies – Detailed Notes

Gene Transfer Technologies – Detailed Notes 1. Definition Gene transfer is the process of introducing foreign DNA or genes into the genome of a target organism or cell. It allows the expression of new traits, study of gene function, and production of therapeutic proteins. Also known as gene delivery or genetic transformation. 2. Principles of Gene Transfer Involves delivery of DNA or RNA into cells or organisms. DNA can be integrated into the host genome or remain episomal (non-integrated). The goal is stable or transient expression of the transferred gene. Key considerations: Vector – vehicle for carrying the gene Target cell – plant, animal, microbial, or human cells Delivery method – physical, chemical, or biological 3. Types of Gene Transfer Gene transfer can be broadly classified into: A. Natural Gene Transfer Occurs in nature between organisms: Transformation: Uptake of naked DNA by bacteria. Transduction: DNA transfer via viruses (bacteriophages). Conjugation: Transfer of plasmi...
Post a Comment
Read more