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Questions and Answers
Which of the following are revision cloning techniques? Select all that apply.
Which of the following are revision cloning techniques? Select all that apply.
Which two cloning techniques are newer cloning techniques?
Which two cloning techniques are newer cloning techniques?
Overlap Extension PCR is used for the introduction of mutations.
Overlap Extension PCR is used for the introduction of mutations.
True
What are the two main enzymes used for Overlap Extension PCR?
What are the two main enzymes used for Overlap Extension PCR?
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What is the main disadvantage of Overlap Extension PCR?
What is the main disadvantage of Overlap Extension PCR?
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Which of the following enzymes is required for QuikChange Mutagenesis?
Which of the following enzymes is required for QuikChange Mutagenesis?
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QuikChange Mutagenesis has fewer steps than Overlap Extension PCR, but it still uses polymerase and ligase.
QuikChange Mutagenesis has fewer steps than Overlap Extension PCR, but it still uses polymerase and ligase.
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What is the main disadvantage of QuikChange Mutagenesis?
What is the main disadvantage of QuikChange Mutagenesis?
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Which enzyme is used in Topo Cloning?
Which enzyme is used in Topo Cloning?
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Topo Cloning uses PCR products generated with Taq DNA polymerase because those products have 3' overhang with A's.
Topo Cloning uses PCR products generated with Taq DNA polymerase because those products have 3' overhang with A's.
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What is the main disadvantage of Topo Cloning?
What is the main disadvantage of Topo Cloning?
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Which enzyme is used in Gibson Assembly?
Which enzyme is used in Gibson Assembly?
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Gibson Assembly can successfully assemble multiple fragments of DNA, regardless of length or end compatibility.
Gibson Assembly can successfully assemble multiple fragments of DNA, regardless of length or end compatibility.
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What is the main disadvantage of Gibson Assembly?
What is the main disadvantage of Gibson Assembly?
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What is the main difference between Gateway Cloning and the traditional restriction digestion methods?
What is the main difference between Gateway Cloning and the traditional restriction digestion methods?
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Gateway Cloning uses site-specific recombination to integrate a fragment of interest into a vector.
Gateway Cloning uses site-specific recombination to integrate a fragment of interest into a vector.
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What is the main disadvantage of Gateway Cloning?
What is the main disadvantage of Gateway Cloning?
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Study Notes
Lecture 07 - BIOT 732 - Genetic Engineering II
- Topic: Cloning Techniques
- Subtopic: Nuclease-mediated DNA editing techniques
- Specific techniques mentioned: ZFN, TALEN
- Learning Outcomes: Describe and distinguish cloning techniques
Cloning Techniques - Revision
- Overlap Extension PCR
- QuikChange Mutagenesis
- Topo Cloning
- Gibson Assembly Cloning
- Gateway Cloning
- Golden Gate Assembly
Cloning Techniques - Overlap Extension PCR (Cell Biology)
- Step 1: Two PCR reactions are performed using flanking primers and mutagenesis primers to create complementary sequences.
- Step 2: The products from step 1 are mixed to combine the flanking regions. A second PCR reaction is then performed to assemble the overlapping fragments creating the desired sequence.
- Application: Introducing mutations
- Needed: Gene of interest, standard enzymes (polymerase, ligase, alkaline phosphatase, restriction enzymes)
- Advantages: Simple techniques, available in many labs, no extra kit cost
- Disadvantages: Long procedure (2-3 days), multiple steps involved
Cloning Techniques - QuikChange Mutagenesis (Cell Biology)
-
Steps:
- Mutant strand synthesis (normal PCR).
- Denature and anneal mutagenic primers with desired mutation in the template DNA.
- Extend and incorporate primers using PfuUltra DNA polymerase
- Dpnl digestion to digest the parental methylated and hemimethylated DNA
- Transformation of mutated molecule to competent cells.
- Application: introducing mutations
- Needed: appropriate gene, Dpnl enzyme
- Advantages: Less steps than overlap extension method, efficient, no extra cost
- Disadvantages: Difficult and inefficient PCR reaction
Cloning Techniques - Topo Cloning (GEI)
- Background: Topoisomerases change the state of DNA supercoiling. Topoisomerase I creates transient single-strand breaks in the phosphate backbone of DNA.
- Mechanism: Topoisomerase I forms temporary ester bonds with DNA, creating transient single-strand breaks and facilitating the reformation of the original phosphodiester bond. Then the enzyme releases from the DNA.
- Ligase-independent ligation: Topo Cloning utilizes topoisomerase I’s ability to form phosphodiester bonds for ligation of newly synthesized DNA molecules.
- PCR products: Taq DNA polymerase generated PCR products have 3′ overhanging A's.
- Application: insertion of PCR products/genes.
- Advantages: Simple technique, fewer steps compared to classical methods, insertion of long fragments possible (specific Topo Kit)
- Disadvantages: high cost for kit, using a special vector, inflexible
New Cloning Techniques - Gibson Assembly® Cloning
- Description: One-step, isothermal reaction
- Enzymatic Activities: Exonuclease creates single-stranded 3' overhangs, polymerase fills in gaps, ligase seals nicks
- End Result: Double-stranded, fully sealed DNA molecule
- Applications: PCR template creation or molecular biology applications
New Cloning Techniques - Gateway Cloning
- Background: Site-specific recombination
- Mechanism: The method uses attP and attB sites on the vector/DNA fragment
- Steps: BP reaction (integration) and LR reaction (excision)
- Advantages: Highly efficient, no restriction enzymes required
- Disadvantages: Potential for large scar sequences, multiple entry clones might be needed for multiple gene insertion into one vector.
New Cloning Techniques - Golden Gate Assembly
- Description: One-step, isothermal cloning method
- Mechanism: Uses Type II restriction enzymes to digest a donor DNA/target DNA that results in compatible overlapping overhangs to be assembled via ligation
- Advantages: One-step cloning procedure and suitable for linking of multiple overlapping fragments in one reaction.
- Disadvantages: Requires the use of enzymes with the correct properties, can be more complex to perform a multi-insert cloning experiment
Nuclease-mediated DNA editing techniques
- Techniques:
- Zinc-Finger Nucleases (ZFNs)
- Transcription Activator-Like Effector Nucleases (TALENs)
Other Notes
- Primer design, PCR amplification steps associated with each cloning technique
- Protocols and components associated with each method.
- Examples and application of each cloning technique
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Description
This quiz focuses on various cloning techniques, including nuclease-mediated DNA editing methods such as ZFN and TALEN. It provides insights into advanced techniques like Overlap Extension PCR and Gibson Assembly, along with their applications and advantages. Test your knowledge on these important aspects of genetic engineering!
