46 Questions
What is the main purpose of Recombinant DNA technology?
To produce DNA via artificial means using protocols like gene cloning and genetic engineering
Which enzymes are essential for constructing Recombinant DNA?
Restriction Enzymes and DNA Ligase
What is the role of a vector in Recombinant DNA technology?
To transfer foreign genetic material into another cell
Why are Type II restriction endonucleases important in Recombinant DNA technology?
They cut DNA molecules internally at specific base pair sequences
What is the process that involves the introduction of rDNA into eukaryotic cells?
Transfection
How are host cells made more permeable to take up exogenous DNA in the process of cell competency?
Treatment with calcium chloride
What technology has allowed for the production of human insulin using bacteria as host cells?
Recombinant DNA technology
What was the method used for cloning and expression of hGH in Chinese hamster ovary (CHO) cells?
Recombinant DNA technology
What did the use of mammalian cells ensure in the production of recombinant human growth hormone (hGH)?
Proper post-translational modifications
What is the process that can occur naturally in some bacteria or be induced in the laboratory, involving the uptake and incorporation of rDNA into the bacterial cell?
Transformation
What is the source of the first identified restriction endonuclease, EcoRI?
Escherichia coli
How do restriction enzymes derive their names?
From the bacterial species they come from
What is the function of sticky ends produced by some restriction enzymes?
Allow fragments to join through complementary base pairing
How do blunt ends differ from sticky ends in DNA fragments?
Blunt ends have no overhangs and make ligation less efficient
How do host cells protect their own DNA from restriction endonucleases?
Through methylation
What are vectors used for in genetic engineering?
To carry and propagate foreign DNA in a host cell
What type of DNA molecules are plasmids?
Circular extrachromosomal DNA molecules used as vectors
Why are bacteriophages used as vectors?
For their large insert size and ease of screening through plaque formation
What distinguishes cosmids from other vectors?
- Cosmids are hybrid vectors that combine plasmid and bacteriophage features, allowing for the cloning of larger DNA fragments.
What is the process that involves the introduction of rDNA into eukaryotic cells?
Transfection
How are host cells made more permeable to take up exogenous DNA in the process of cell competency?
Treatment with calcium chloride
What was the method used for cloning and expression of hGH in Chinese hamster ovary (CHO) cells?
Electroporation
What is the role of recombinant DNA technology in insulin production?
Allows production of human insulin using bacteria as host cells
What is the source of recombinant human growth hormone (hGH) prior to the development of recombinant DNA technology?
Extracted from cadaveric pituitary glands
What is the process that can occur naturally in some bacteria or be induced in the laboratory, involving the uptake and incorporation of rDNA into the bacterial cell?
Transformation
What technology has allowed for proper post-translational modifications, such as glycosylation, in the production of recombinant human growth hormone (hGH)?
Recombinant DNA technology
What is the method for making cells competent by creating temporary pores on the cell membrane?
Heat shock
What is transferred from one bacterium to another through a viral vector?
rDNA
What process involves introducing foreign genetic material into cells?
Insertion into Host
What is the main purpose of Recombinant DNA technology?
To transfer foreign genetic material into another cell using vectors
What are Type II restriction endonucleases important for in Recombinant DNA technology?
Cutting DNA molecules internally at specific base pair sequences
What is the function of a vector in Recombinant DNA technology?
To transfer foreign genetic material into another cell
What is the role of a host cell in Recombinant DNA technology?
To carry out the genetic modification
What is the purpose of screening/identifying host cell colonies in Recombinant DNA technology?
To identify the specific colony containing the target gene
What is a vector in the context of Recombinant DNA technology?
A vehicle to transfer foreign genetic material into another cell
What is the function of restriction enzymes in Recombinant DNA technology?
To cut DNA molecules internally at specific base pair sequences
What is the basic principle of constructing recombinant DNA?
Joining insert DNA to a cloning vector and transferring it into a host cell
Why must both source DNA and cloning vector be consistently cut into discrete and reproducible fragments in Recombinant DNA technology?
To prevent random integration of foreign genes into host cells' genomes
What is the purpose of transferring each DNA construct into a host cell in Recombinant DNA technology?
To maintain it and allow for expression of target genes
What is the maximum insert size of Bacterial Artificial Chromosomes (BACs)?
300 kb
What type of DNA molecules are plasmids?
Circular extrachromosomal DNA molecules
Why are bacteriophages used as vectors?
Due to their large insert size and ease of screening through plaque formation
How do restriction enzymes derive their names?
From the bacterial species they come from
What is the function of sticky ends produced by some restriction enzymes?
Help in DNA cloning by allowing fragments to join through complementary base pairing
What distinguishes cosmids from other vectors?
They combine plasmid and bacteriophage features, allowing for the cloning of larger DNA fragments
What is the source of the first identified restriction endonuclease, EcoRI?
E. coli
Study Notes
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Escherichia coli (E.coli) is the source of the first identified restriction endonuclease, named EcoRI.
-
Restriction enzymes derive their names from the bacterial species they come from.
-
Restriction enzymes work by recognizing and binding to specific palindromic sequences in DNA, cutting between G and A residues.
-
Sticky ends are produced by some restriction enzymes, which leave single-stranded overhangs and help in DNA cloning by allowing fragments to join through complementary base pairing.
-
Blunt ends result when a restriction enzyme cuts both strands at the same position, leaving no overhangs and making ligation less efficient and more complex.
-
Host cells protect their own DNA from restriction endonucleases through methylation.
-
Vectors are DNA molecules used to carry and propagate foreign DNA in a host cell, and have specific sites for insertion of target DNA, origins of replication, selectable markers, and multiple cloning sites.
-
Plasmids are circular extrachromosomal DNA molecules used as vectors, which can be found in bacteria, eukaryotes, and archaea, and are small, easy to detect due to antibiotic resistance genes, and have a limited insert size.
-
Bacteriophages are used as vectors due to their large insert size and ease of screening through plaque formation.
-
Cosmids are hybrid vectors that combine plasmid and bacteriophage features, allowing for the cloning of larger DNA fragments.
-
Bacterial Artificial Chromosomes (BACs) and Yeast Artificial Chromosomes (YACs) are large DNA constructs used for cloning, with BACs having a bacterial origin and a maximum insert size of 300 kb, and YACs having a yeast origin and a maximum insert size of 2000 kb.
-
Recombinant DNA is introduced into a host cell for replication and expression, with bacteria, yeast, and mammalian cells being used depending on the desired product.
-
Escherichia coli (E.coli) is the source of the first identified restriction endonuclease, named EcoRI.
-
Restriction enzymes derive their names from the bacterial species they come from.
-
Restriction enzymes work by recognizing and binding to specific palindromic sequences in DNA, cutting between G and A residues.
-
Sticky ends are produced by some restriction enzymes, which leave single-stranded overhangs and help in DNA cloning by allowing fragments to join through complementary base pairing.
-
Blunt ends result when a restriction enzyme cuts both strands at the same position, leaving no overhangs and making ligation less efficient and more complex.
-
Host cells protect their own DNA from restriction endonucleases through methylation.
-
Vectors are DNA molecules used to carry and propagate foreign DNA in a host cell, and have specific sites for insertion of target DNA, origins of replication, selectable markers, and multiple cloning sites.
-
Plasmids are circular extrachromosomal DNA molecules used as vectors, which can be found in bacteria, eukaryotes, and archaea, and are small, easy to detect due to antibiotic resistance genes, and have a limited insert size.
-
Bacteriophages are used as vectors due to their large insert size and ease of screening through plaque formation.
-
Cosmids are hybrid vectors that combine plasmid and bacteriophage features, allowing for the cloning of larger DNA fragments.
-
Bacterial Artificial Chromosomes (BACs) and Yeast Artificial Chromosomes (YACs) are large DNA constructs used for cloning, with BACs having a bacterial origin and a maximum insert size of 300 kb, and YACs having a yeast origin and a maximum insert size of 2000 kb.
-
Recombinant DNA is introduced into a host cell for replication and expression, with bacteria, yeast, and mammalian cells being used depending on the desired product.
Learn about the fundamentals of recombinant DNA technology, including gene cloning, genetic engineering, and molecular cloning. Understand how DNA can be produced through artificial means and how recombinant DNA is constructed from different sources using restriction enzymes, DNA ligase, and vectors.
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