Genetic Engineering II - Cloning Techniques

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Questions and Answers

Which of the following are revision cloning techniques? Select all that apply.

  • QuikChange Mutagenesis (correct)
  • Gateway Cloning Experiment
  • Overlap Extension PCR (correct)
  • Gibson Assembly® Cloning
  • Topo Cloning (correct)
  • Golden Gate Assembly

Which two cloning techniques are newer cloning techniques?

  • Golden Gate Assembly (correct)
  • Overlap Extension PCR
  • QuikChange Mutagenesis
  • Gateway Cloning Experiment (correct)
  • Gibson Assembly® Cloning
  • Topo Cloning

Overlap Extension PCR is used for the introduction of mutations.

True (A)

What are the two main enzymes used for Overlap Extension PCR?

<p>Polymerase and Ligase</p> Signup and view all the answers

What is the main disadvantage of Overlap Extension PCR?

<p>It's a long procedure, requiring 2-3 days.</p> Signup and view all the answers

Which of the following enzymes is required for QuikChange Mutagenesis?

<p>DpnI (C)</p> Signup and view all the answers

QuikChange Mutagenesis has fewer steps than Overlap Extension PCR, but it still uses polymerase and ligase.

<p>False (B)</p> Signup and view all the answers

What is the main disadvantage of QuikChange Mutagenesis?

<p>It is a very inefficient PCR reaction.</p> Signup and view all the answers

Which enzyme is used in Topo Cloning?

<p>Topoisomerase I (A)</p> Signup and view all the answers

Topo Cloning uses PCR products generated with Taq DNA polymerase because those products have 3' overhang with A's.

<p>True (A)</p> Signup and view all the answers

What is the main disadvantage of Topo Cloning?

<p>The kits are very expensive, especially for eukaryotic expression, and the vector is inflexible.</p> Signup and view all the answers

Which enzyme is used in Gibson Assembly?

<p>Exonuclease (B), Ligase (C)</p> Signup and view all the answers

Gibson Assembly can successfully assemble multiple fragments of DNA, regardless of length or end compatibility.

<p>True (A)</p> Signup and view all the answers

What is the main disadvantage of Gibson Assembly?

<p>The kit is moderately expensive, costing around 120 Euros for 10 reactions without competent cells.</p> Signup and view all the answers

What is the main difference between Gateway Cloning and the traditional restriction digestion methods?

<p>Gateway cloning does not require restriction enzymes. (A)</p> Signup and view all the answers

Gateway Cloning uses site-specific recombination to integrate a fragment of interest into a vector.

<p>True (A)</p> Signup and view all the answers

What is the main disadvantage of Gateway Cloning?

<p>The process leaves behind a 25 bp junk sequence (scar).</p> Signup and view all the answers

Flashcards

Overlap Extension PCR

A method for introducing mutations into a gene of interest by using PCR with overlapping primers that contain the desired mutations.

QuikChange Mutagenesis

A technique for introducing point mutations into a gene of interest using PCR with primers containing the desired mutations, followed by digestion with DpnI to remove the original methylated template.

Topo Cloning

A technique that allows the ligation of PCR-generated DNA fragments into a vector without the need for restriction enzymes. It exploits the ability of Topoisomerase I to form a covalent bond with DNA and then ligate the DNA fragment into the vector.

Gibson Assembly Cloning

A method for cloning multiple DNA fragments in a single tube using three enzymes: an exonuclease to create overhangs, a polymerase to fill in gaps, and a ligase to seal nicks.

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Golden Gate Assembly

A method for cloning multiple DNA fragments by using Type IIS restriction enzymes that recognize a non-palindromic sequence and cleave outside the recognition site, producing unique overhangs that can be ligated together in a single reaction.

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Zinc-Finger Nucleases (ZFNs)

Programmable DNA endonucleases that consist of a zinc finger protein (ZFP) fused to the cleavage domain of the FokI restriction enzyme.

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Transcription Activator-Like Effectors (TALEs)

A family of proteins that bind to DNA at specific sites within the genome.

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Transcription Activator-Like Effector Nucleases (TALENs)

Artificially engineered restriction enzymes used for genome editing. They consist of a TALE DNA-binding domain fused to the FokI nuclease.

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Type IIS Restriction Enzymes

A type of enzyme that cleaves DNA outside of its recognition sequence, leaving unique overhangs that can be used for cloning.

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Entry Clone

A plasmid that contains the gene of interest between two attL sites.

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Donor Vector

A plasmid that allows for the recombination of a gene of interest between attB sites and attP sites, resulting in an entry clone.

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Destination Vector

A plasmid that contains two attR sites, allowing for the insertion of a gene of interest from an entry clone.

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LR Reaction

The process of using the Gateway LR Clonase enzyme to transfer a gene of interest from an entry clone to a destination vector.

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BP Reaction

The process of using the Gateway BP Clonase enzyme to recombine a gene of interest with a donor vector, creating an entry clone.

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Multisite Gateway Cloning

A technique for efficiently inserting multiple DNA fragments into a single destination vector by using four different att sites.

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Golden Gate Cloning

A method for assembling two or more DNA molecules together in a one-pot, single-step reaction using Type II restriction enzymes.

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Domestication

The process of removing unwanted Type IIS restriction enzyme recognition sites from a DNA molecule, typically a vector.

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Fusion Site

A short sequence of DNA that is recognized by a Type IIS restriction enzyme.

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ccdB Gene

A suicide gene that is lethal to bacteria that do not contain a compatible counter-selection marker.

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Selection Marker

A marker that allows for the selection of cells that contain a specific gene of interest.

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Non-Homologous End Joining (NHEJ)

A type of DNA repair that replaces damaged or missing DNA sequences with random sequences.

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Homologous Recombination (HR)

A type of DNA repair that uses a homologous template to repair damaged DNA, resulting in accurate repair.

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Targeted Mutagenesis

A process that can lead to a mutation in a gene, often caused by the insertion or deletion of a base pair during DNA repair.

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Targeted Gene Editing

The ability of a gene to be modified or edited.

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Targeted Gene Replacement

A process that can lead to the replacement of a gene with a different version, often used for gene therapy.

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Restriction Cloning

A type of cloning where a DNA fragment is inserted into a vector by using restriction enzymes to cut the DNA at specific sites.

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Genomic DNA Library

A set of DNA fragments that represent the entire genome of an organism, used for research and genetic analysis.

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Reverse Genetics

A scientific approach that begins with a gene of interest and aims to study its function by modifying or deleting it.

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Transgenic Cell

A type of cell that is genetically modified to express a specific gene of interest.

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Transformation

The process of introducing foreign DNA into a cell.

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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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Lecture 07 - BIOT 732 Genetic Engineering II Lecture Notes PDF

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