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Questions and Answers
What is the characteristic of type 111 restriction endonucleases in terms of ATP requirement?
What is the characteristic of type 111 restriction endonucleases in terms of ATP requirement?
What is the typical length of the recognition sequence for restriction endonucleases?
What is the typical length of the recognition sequence for restriction endonucleases?
Where do type 11 restriction endonucleases cleave the DNA?
Where do type 11 restriction endonucleases cleave the DNA?
What is the term for the unpaired strands left at the ends of DNA fragments after cleavage by restriction endonucleases?
What is the term for the unpaired strands left at the ends of DNA fragments after cleavage by restriction endonucleases?
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What is the significance of the first three letters in the name of a restriction enzyme?
What is the significance of the first three letters in the name of a restriction enzyme?
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What is the characteristic of the ends of DNA fragments after cleavage by certain restriction endonucleases?
What is the characteristic of the ends of DNA fragments after cleavage by certain restriction endonucleases?
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What is a characteristic of most restriction enzymes?
What is a characteristic of most restriction enzymes?
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What is the function of DNA ligase in gene cloning?
What is the function of DNA ligase in gene cloning?
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What is the energy source required for DNA ligase to function?
What is the energy source required for DNA ligase to function?
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What is one of the medical applications of gene cloning?
What is one of the medical applications of gene cloning?
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What is the purpose of gene therapy?
What is the purpose of gene therapy?
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What is one of the agricultural applications of gene cloning?
What is one of the agricultural applications of gene cloning?
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What is the main goal of recombinant DNA technology?
What is the main goal of recombinant DNA technology?
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What is the purpose of introducing a target gene into a plasmid?
What is the purpose of introducing a target gene into a plasmid?
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What is a characteristic of a cloning vector?
What is a characteristic of a cloning vector?
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What is the purpose of a marker gene in a cloning vector?
What is the purpose of a marker gene in a cloning vector?
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What is the process called when a plasmid is introduced into bacteria?
What is the process called when a plasmid is introduced into bacteria?
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Why are antibiotics used in a typical cloning experiment?
Why are antibiotics used in a typical cloning experiment?
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What is the purpose of restriction endonuclease enzyme in gene technology?
What is the purpose of restriction endonuclease enzyme in gene technology?
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What is the function of DNA methylase in bacteria?
What is the function of DNA methylase in bacteria?
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What is the result of the action of Type 1 restriction endonuclease on DNA?
What is the result of the action of Type 1 restriction endonuclease on DNA?
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What is the purpose of DNA ligase in gene technology?
What is the purpose of DNA ligase in gene technology?
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What is the natural function of restriction endonuclease in bacteria?
What is the natural function of restriction endonuclease in bacteria?
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What is the purpose of the fourth step in the gene technology process?
What is the purpose of the fourth step in the gene technology process?
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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.