BIOT 732: Genetic Engineering II

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Questions and Answers

What are the three main disadvantages of Gateway cloning?

(i) The cloning vector can be linearized by any restriction endonuclease or by any combination of two restriction endonucleases displaying unique site(s) at the desired locations within the vector sequence. (ii) Double-digestion of vector DNA with two restriction endonucleases is the best approach to reduce the uncut vector background. (iii) Some restriction endonucleases cannot efficiently digest supercoiled DNA and thus may leave behind different amounts of uncut vector DNA. If not gel-purified, the uncut vector is transformable and will show up after transformation of the Gibson Assembly reaction, thereby, reducing the overall fraction of recombinant clones.

(i) the associated two-step (BP and LR recombination reactions) cloning process is labor-intensive and time-consuming (ii) the recombination site leaves a 25-bp unwanted junk sequence (scar) (iii) The end result is a double-stranded fully sealed DNA molecule that can serve as template for PCR, or a variety of other molecular biology applications, including direct transformation. (iv) The method has been successfully used by Gibson's group and others to assemble oligonucleotides, DNA with varied overlaps (15–80 bp) and fragments hundreds of kilobases long.

(i) The associated two-step (BP and LR recombination reactions) cloning process is labor-intensive and time-consuming (ii) the method is not as fast and efficient as other cloning methods. (iii) The assembly of multiple fragments is relatively inefficient (iv) The commercial enzyme mixes available for this method are expensive, especially for laboratories in developing countries.

(i) the associated two-step (BP and LR recombination reactions) cloning process is labor-intensive and time-consuming (ii) the recombination site leaves a 25-bp unwanted junk sequence (scar) (iii) the assembly of multiple fragments is relatively inefficient (iv) the commercial enzyme mixes available for this method are expensive, especially for laboratories in developing countries. (correct)

What are the benefits of using Gateway cloning?

Large libraries of cloned and sequenced gene fragments are available for purchase, thus allowing rapid manipulation of genes. (correct)

Large scar size in the resulting DNA fragment, resulting in increased gene expression in the host bacteria.

The process is highly efficient, enabling the linking of multiple overlapping DNA fragments in a single isothermal reaction, thus saving time and resources.

The same LR reaction can be applied to several different clones of interest, allowing for a more flexible and efficient workflow. (correct)

What happens when the phage lambda senses that the bacteria is under stress?

It will begin its 'lytic growth cycle' to destroy the host bacteria.

It will use site-specific recombination to quickly replicate itself and release numerous progeny phage particles.

It will integrate itself into the bacterial genome and begin its lysogenic phase, becoming dormant.

It will start the process of excision to remove its DNA from the bacterial chromosome, and then begin its lytic growth cycle to infect other bacteria. (correct)

What is the name of the mechanism used by phage lambda to integrate its genome into E. coli?

Site-specific recombination Signup and view all the answers

Gateway cloning does not require the use of restriction enzymes.

True Signup and view all the answers

In Golden Gate Assembly, the ______ site is non-palindromic, ranging from 4 to 7 nucleotides.

recognition Signup and view all the answers

What is the purpose of using TALENs in genome engineering?

To create gene knockouts or introduce specific DNA sequence modifications. Signup and view all the answers

Zinc-finger nucleases (ZFN) are the first widely used programmable DNA binding protein system.

True Signup and view all the answers

Match the following terms with their relevant definitions:

TALENs = A special class of proteins that can bind DNA ZFNs = Artificial enzymes that target and cut DNA at specific sequences Golden Gate Assembly = A method of cloning that uses Type IIS restriction enzymes to join DNA fragments in a single reaction. Gateway cloning = A method of cloning that relies on site-specific recombination reactions and utilizes att sites. Signup and view all the answers

What is the name of the enzyme used in TALENs that produces a double-stranded break in DNA?

FokI Signup and view all the answers

What is the main advantage of using Golden Gate Assembly?

The use of 'scarless' or 'seamless' cloning, which eliminates undesired nucleotides in the final construct. Signup and view all the answers

In Golden Gate Assembly, the enzyme recognition sites in a destination vector are oriented with an 'inward' orientation.

False Signup and view all the answers

Study Notes

Lecture 07 - BIOT 732: Genetic Engineering II

Topic: Cloning Techniques
Subtopic: Nuclease-mediated DNA editing Techniques
Learning Outcome: Describe and distinguish between the following cloning techniques.
Revision: Overlap Extension PCR, QuikChange Mutagenesis, Topo Cloning
New Techniques: Gibson Assembly® Cloning, Gateway Cloning Experiment, Golden Gate Assembly, ZFN, TALEN

Learning Outcome

Describe and distinguish between cloning techniques

Revision: Cloning Techniques

Overlap Extension PCR
- Application: Introduction of Mutations
- Needs: Gene of interest in own vector, standard enzymes (polymerase, ligase, alkaline phosphatase, restriction enzymes)
- Advantages: No sophisticated techniques, several steps available in every lab. No extra cost for kits.
- Disadvantages: Long procedure with many steps (2-3 days)
QuikChange Mutagenesis
- Application: Introduction of a Mutation
- Needs: Gene of interest in own vector, Enzyme (Dpnl).
- Advantages: Less steps than overlap extension method, quicker. No sophisticated techniques. No extra cost for kits
- Disadvantages: Difficult and very inefficient PCR reaction, random mutation inside vector inserted during PCR remains unnoticed.
Topo Cloning
- Background: Topoisomerases change the state of supercoiling of DNA to decrease gene activity, introducing temporary single-strand (Type I) or double-strand (Type II) breaks in the phosphate backbone
- Mechanism: Topoisomerase action transiently forms an ester bond between a tyrosine residue of the enzyme and the DNA molecule.
- Ligase-independent ligation of PCR products: Topoisomerase-I forms phosphodiester bonds which can be used for ligation of DNA molecules
- PCR products generated with Taq DNA polymerase have 3' overhanging As

New Cloning Techniques

Gibson Assembly® Cloning
- Introduced by NEB by Dr. Daniel Gibson
- Allows for successful assembly of multiple DNA fragments regardless of fragment length or end compatibility.
- Efficiently joins multiple overlapping DNA fragments in a single-tube isothermal reaction
- Method includes exonuclease, DNA polymerase and DNA ligase
- Method is used by Gibson's group and others to assemble oligonucleotides, DNA with varied overlaps (15–80 bp) and fragments hundreds of kilobases long
Gateway Cloning
- Highly efficient alternative to restriction cloning.
- Uses a two-step recombination process with attachment sites attL and attB.
- Based on site-specific recombination machinery used by phage (lambda) -att sites = attachment site of ~20 nts
- If phage senses bacteria stress, it excises itself, and phage DNA is removed from bacterial chromosome
  - Gateway cloning moves elements through a BP and LR reaction that are reversible.
Golden Gate Assembly
- One-pot, one-step cloning
- Method utilizes Type IIS restriction enzymes (e.g., Bsal).
- Method yields ordered assembly of vector and DNA fragments
- Two steps: Type IIS restriction enzyme digestion and DNA ligation.
Nuclease-mediated DNA editing techniques
- Zinc-Finger Nucleases (ZFNs)
- Transcription Activator-Like Effector Nucleases (TALENS)

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Description

Test your knowledge on advanced cloning techniques covered in BIOT 732: Genetic Engineering II. This quiz explores various methods including Overlap Extension PCR and Gibson Assembly®. Distinguish between these techniques and understand their applications and limitations.