Restriction Endonucleases in Molecular Biology

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Questions and Answers

What is the characteristic of type 111 restriction endonucleases in terms of ATP requirement?

They require ATP energy only in specific conditions

They require ATP energy (correct)

They sometimes require ATP energy

They do not require ATP energy

What is the typical length of the recognition sequence for restriction endonucleases?

11 to 15 base pairs

4 to 6 base pairs (correct)

7 to 10 base pairs

2 to 3 base pairs

Where do type 11 restriction endonucleases cleave the DNA?

Within the recognition sequence itself (correct)

At the opposing phosphodiester bonds

At the ends of the DNA molecule

About 25 bp from the recognition sequence

What is the term for the unpaired strands left at the ends of DNA fragments after cleavage by restriction endonucleases?

Sticky ends Signup and view all the answers

What is the significance of the first three letters in the name of a restriction enzyme?

They represent the species in which the enzyme was found Signup and view all the answers

What is the characteristic of the ends of DNA fragments after cleavage by certain restriction endonucleases?

They can have either sticky or blunt ends Signup and view all the answers

What is a characteristic of most restriction enzymes?

They recognize a single restriction site Signup and view all the answers

What is the function of DNA ligase in gene cloning?

To join DNA fragments covalently Signup and view all the answers

What is the energy source required for DNA ligase to function?

ATP Signup and view all the answers

What is one of the medical applications of gene cloning?

Production of recombinant human insulin Signup and view all the answers

What is the purpose of gene therapy?

To remove a defective gene and replace it with a healthy one Signup and view all the answers

What is one of the agricultural applications of gene cloning?

Production of pest-resistant plants Signup and view all the answers

What is the main goal of recombinant DNA technology?

To combine DNA from two distinct sources Signup and view all the answers

What is the purpose of introducing a target gene into a plasmid?

To make many identical copies of a piece of DNA Signup and view all the answers

What is a characteristic of a cloning vector?

It must be self-replicating inside a host cell Signup and view all the answers

What is the purpose of a marker gene in a cloning vector?

To identify recombinant cells Signup and view all the answers

What is the process called when a plasmid is introduced into bacteria?

Transformation Signup and view all the answers

Why are antibiotics used in a typical cloning experiment?

To select bacteria carrying the plasmid Signup and view all the answers

What is the purpose of restriction endonuclease enzyme in gene technology?

To cut DNA of the gene and the vector at a specific site Signup and view all the answers

What is the function of DNA methylase in bacteria?

To protect the host's cell DNA from digestion Signup and view all the answers

What is the result of the action of Type 1 restriction endonuclease on DNA?

Cuts DNA at random sites that can be more than 1000 base pair from the recognition sequence Signup and view all the answers

What is the purpose of DNA ligase in gene technology?

To join the gene of interest with the vector Signup and view all the answers

What is the natural function of restriction endonuclease in bacteria?

To recognize and cleave foreign DNA Signup and view all the answers

What is the purpose of the fourth step in the gene technology process?

Selection of transformed host cells and identification of the clone Signup and view all the answers

Study Notes

Characteristics of Restriction Enzymes

Most restriction enzymes recognize a single restriction site.
The restriction site is recognized without regard to the source of DNA.

DNA Ligase

DNA ligase is an enzyme that covalently links adjacent 3'-oH and 5'-po4 groups.
The joining of DNA fragments requires energy, which in humans is provided by ATP.

Applications of Gene Cloning

Medical applications: gene cloning plays a crucial role in synthesizing vitamins, hormones, and antibiotics.
Medical applications: produces recombinant human insulin (Humulin) in E. coli for diabetic patients.
Agriculture applications: cloning is used to create pest-resistant plants.
Gene therapy: involves replacing defective genes with new healthy ones, e.g., treating leukemia and sickle cell anemia.

Steps in Gene Cloning

Isolation of DNA fragments to be cloned.
Insertion of isolated DNA into a suitable vector to form recombinant DNA.
Introduction of recombinant DNA into a suitable host organism.
Selection of transformed host cells and identification of the clone containing the gene of interest.
Multiplication/expression of the introduced gene in the host.
Isolation of multiple gene copies/protein expressed by the gene.
Purification of the isolated gene copy/protein.

Enzymes Used in Gene Technology

Restriction endonuclease enzyme (cut): cuts DNA at specific sites forming sticky ends.
DNA ligase (paste): joins the gene of interest with the vector.

Types of Restriction Endonucleases

Type 1: cleaves DNA at random sites, often >1000 bp from the recognition sequence.
Type 2: cleaves DNA within the recognition sequence itself and requires no ATP.
Type 3: cleaves DNA about 25 bp from the recognition sequence and requires ATP.

Characteristics of Cloning Vectors

Self-replicating inside host cells.
Possess a unique restriction site for RE enzymes.
Introduction of donor DNA fragment must not interfere with replication property of the vector.
Possess a marker gene for later identification of recombinant cells (usually an antibiotic resistance gene).
Easily isolated from host cells.

Naming Restriction Enzymes

Most restriction enzymes are named after the species in which they were found.
The first three letters in the name of each restriction enzyme stand for the bacterial species of origin.

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Description

This quiz covers the characteristics of type I, II, and III restriction endonucleases, including their mechanisms, energy requirements, and recognition sequences. Understand the differences between these types and how they cleave DNA.