Podcast
Questions and Answers
What is the primary function of a Bacterial Artificial Chromosome (BAC)?
What is the primary function of a Bacterial Artificial Chromosome (BAC)?
Expression vectors can produce proteins that make up 40% of a cell's total protein.
Expression vectors can produce proteins that make up 40% of a cell's total protein.
True
What gene expression system is primarily used in expression vectors?
What gene expression system is primarily used in expression vectors?
Phage T7 RNA polymerase
BACs can accommodate inserts of up to __________ kbp.
BACs can accommodate inserts of up to __________ kbp.
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Match the following vector types with their applications:
Match the following vector types with their applications:
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Which component is essential for the transcriptional control in an expression vector?
Which component is essential for the transcriptional control in an expression vector?
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Bacterial Artificial Chromosomes can exist in multiple copies within a single bacterium.
Bacterial Artificial Chromosomes can exist in multiple copies within a single bacterium.
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What is the role of the lac Z′ gene in BACs?
What is the role of the lac Z′ gene in BACs?
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What is the primary function of reverse transcriptase?
What is the primary function of reverse transcriptase?
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Plasmids with stringent control can replicate independently of the host chromosome.
Plasmids with stringent control can replicate independently of the host chromosome.
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What enzyme is used to synthesize the second strand of DNA after cDNA synthesis?
What enzyme is used to synthesize the second strand of DNA after cDNA synthesis?
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The lowercase 'p' in plasmid names, such as pBR322, stands for __________.
The lowercase 'p' in plasmid names, such as pBR322, stands for __________.
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Match the following types of vectors with their characteristics:
Match the following types of vectors with their characteristics:
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What is the role of IPTG in the cloning process?
What is the role of IPTG in the cloning process?
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Bacteriophage lambda can only grow lytically and cannot insert its DNA into the host genome.
Bacteriophage lambda can only grow lytically and cannot insert its DNA into the host genome.
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What color do colonies appear when they have undergone successful insertion of foreign DNA using X-gal?
What color do colonies appear when they have undergone successful insertion of foreign DNA using X-gal?
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The genome of bacteriophage lambda is approximately ______ kbp of linear dsDNA.
The genome of bacteriophage lambda is approximately ______ kbp of linear dsDNA.
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Match the following terms with their definitions:
Match the following terms with their definitions:
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Which of the following statements about bacteriophage lambda is true?
Which of the following statements about bacteriophage lambda is true?
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Bacteriophages are less efficient than plasmids for introducing foreign DNA into bacteria.
Bacteriophages are less efficient than plasmids for introducing foreign DNA into bacteria.
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What happens when lambda produces lytic growth?
What happens when lambda produces lytic growth?
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The chromogenic substrate used to detect functional β-galactosidase is called ______.
The chromogenic substrate used to detect functional β-galactosidase is called ______.
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Which of the following correctly identifies the process of ligating cloned DNA?
Which of the following correctly identifies the process of ligating cloned DNA?
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What is the advantage of using double-restriction in cloning?
What is the advantage of using double-restriction in cloning?
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Electroporation creates permanent holes in the bacterial cell membrane.
Electroporation creates permanent holes in the bacterial cell membrane.
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What are the three types of bacterial cells after transformation?
What are the three types of bacterial cells after transformation?
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The enzyme β-galactosidase is active only in E. coli that have plasmids without any cloned DNA within the __________.
The enzyme β-galactosidase is active only in E. coli that have plasmids without any cloned DNA within the __________.
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Match the cloning method to its description:
Match the cloning method to its description:
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Why is directional cloning particularly important in expression systems?
Why is directional cloning particularly important in expression systems?
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The lac Z gene is responsible for the production of an inactive form of β-galactosidase.
The lac Z gene is responsible for the production of an inactive form of β-galactosidase.
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What role does the lac Z' gene play in recombinant DNA technology?
What role does the lac Z' gene play in recombinant DNA technology?
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Bacterial transformation involves making the bacterial membrane __________ to allow plasmid entry.
Bacterial transformation involves making the bacterial membrane __________ to allow plasmid entry.
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Which method is NOT commonly used for bacterial transformation?
Which method is NOT commonly used for bacterial transformation?
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Study Notes
RNA Dependent-DNA Polymerase (Reverse Transcriptase)
- Reverse transcriptase (RT) synthesizes single-stranded DNA from an mRNA template.
- Like regular DNA polymerases, RT needs a primer to start.
- RT possesses 5'→3' exonuclease activity and 3'→5' exonuclease activity, specifically degrading RNA in DNA-RNA hybrid molecules.
- RT extracted from RNA tumor viruses is often used to convert mRNA to dsDNA, for insertion into prokaryotic vectors.
cDNA Synthesis
- First, cDNA is synthesized.
- Next, the RNA is degraded by alkali or ribonuclease H.
- A second strand of cDNA is synthesized using the Klenow fragment of DNA polymerase I or RT itself.
- The cDNA strand acts as a primer and template, forming a hairpin structure.
- Crucially, RT has no proofreading ability.
Cloning and Expression Vectors
Plasmid Vectors
- Plasmids are named using a system of uppercase letters and numbers (e.g. pBR322). The "p" denotes plasmid.
- Some early plasmids had low copy numbers (one or two copies per cell), while others, like pUC18 and pUC19, have high copy numbers (>500 copies per cell).
- High copy number plasmids replicate independently of the host chromosome (relaxed control), whereas stringent control plasmids' replication is tied to the host chromosome.
- An ideal plasmid vector has an origin of replication (ori), selection marker(s) for identifying successful host cells (e.g antibiotic resistance), a multiple cloning site (MCS) for inserting foreign DNA, and efficient extraction from the host cell.
- Techniques like single or twin antibiotic resistance and blue-white screening aid in selecting successful vectors.
Other Vector Types
- Bacteriophage vectors, like lambda (λ), can replicate within bacterial cells, facilitating the insertion of more extensive DNA fragments (>10kbp).
- Cosmids combine the benefits of plasmid (easy propagation) and phage (high capacity) vectors. Cosmids have a cos site, enabling packaging into phage heads, suitable for carrying larger DNA fragments (33-47 kbp).
- Bacterial artificial chromosomes (BACs) are based on the fertility plasmid (F plasmid) allowing the insertion of large DNA fragments (>50 kbp).
- Yeast artificial chromosomes (YACs) can accommodate exceptionally large inserts (100-1000 kbp).
Expression Vectors
- Expression vectors are designed for expressing a target gene at high levels. Typically, they contain a promoter to initiate transcription.
- They use efficient T7 RNA polymerase systems or other protein expression strategies.
- Cloning a target gene into the vector allows its expression in bacterial hosts, which can yield large amounts of the encoded protein.
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Description
This quiz covers the concepts of RNA dependent-DNA polymerase, specifically reverse transcriptase, and its function in synthesizing cDNA. It discusses the processes of cDNA synthesis, the role of exonuclease activity, and the use of plasmid vectors in cloning. Test your knowledge on these essential molecular biology techniques.