RNA Dependent-DNA Polymerase and cDNA Synthesis
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Questions and Answers

What is the primary function of a Bacterial Artificial Chromosome (BAC)?

  • To express proteins in high quantities
  • To clone large DNA sequences (correct)
  • To insert genes into viruses
  • To create plasmids for antibiotic resistance

    • 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?

    Phage T7 RNA polymerase

    BACs can accommodate inserts of up to __________ kbp.

    <p>300</p> Signup and view all the answers

    Match the following vector types with their applications:

    <p>BACs = Cloning large DNA sequences Expression vectors = High-level protein synthesis Cosmids = Cloning medium-sized DNA fragments Selectable markers = Identifying successful clones</p> Signup and view all the answers

    Which component is essential for the transcriptional control in an expression vector?

    <p>Promoter sequence</p> Signup and view all the answers

    Bacterial Artificial Chromosomes can exist in multiple copies within a single bacterium.

    <p>False</p> Signup and view all the answers

    What is the role of the lac Z′ gene in BACs?

    <p>Selection marker</p> Signup and view all the answers

    What is the primary function of reverse transcriptase?

    <p>Synthesize DNA from mRNA</p> Signup and view all the answers

    Plasmids with stringent control can replicate independently of the host chromosome.

    <p>False</p> Signup and view all the answers

    What enzyme is used to synthesize the second strand of DNA after cDNA synthesis?

    <p>Klenow fragment of DNA polymerase I or reverse transcriptase</p> Signup and view all the answers

    The lowercase 'p' in plasmid names, such as pBR322, stands for __________.

    <p>plasmid</p> Signup and view all the answers

    Match the following types of vectors with their characteristics:

    <p>Bacteriophage Lambda = Can accommodate large DNA fragments Cosmids = Hybrid vectors between plasmids and bacteriophages Bacterial Artificial Chromosomes (BACs) = Used for cloning large pieces of DNA Expression Vectors = Directed towards protein production using host machinery</p> Signup and view all the answers

    What is the role of IPTG in the cloning process?

    <p>It is unnecessary for all laboratory strains.</p> Signup and view all the answers

    Bacteriophage lambda can only grow lytically and cannot insert its DNA into the host genome.

    <p>False</p> Signup and view all the answers

    What color do colonies appear when they have undergone successful insertion of foreign DNA using X-gal?

    <p>white</p> Signup and view all the answers

    The genome of bacteriophage lambda is approximately ______ kbp of linear dsDNA.

    <p>48.5</p> Signup and view all the answers

    Match the following terms with their definitions:

    <p>Bacteriophage = A virus that infects bacteria and can be used as a cloning vector Lytic growth = A phase where the virus makes copies of its genome and lyses the host Selectable marker = A gene that allows for the identification of genetically modified organisms Cloning vector = A DNA molecule used to carry foreign DNA into a host cell</p> Signup and view all the answers

    Which of the following statements about bacteriophage lambda is true?

    <p>It produces up to 52 kbp of DNA in its head.</p> Signup and view all the answers

    Bacteriophages are less efficient than plasmids for introducing foreign DNA into bacteria.

    <p>False</p> Signup and view all the answers

    What happens when lambda produces lytic growth?

    <p>It makes copies of its genome and lyses the host cell.</p> Signup and view all the answers

    The chromogenic substrate used to detect functional β-galactosidase is called ______.

    <p>X-gal</p> Signup and view all the answers

    Which of the following correctly identifies the process of ligating cloned DNA?

    <p>The joining of DNA fragments with enzymes.</p> Signup and view all the answers

    What is the advantage of using double-restriction in cloning?

    <p>Reduces the risk of foreign DNA being inserted backward</p> Signup and view all the answers

    Electroporation creates permanent holes in the bacterial cell membrane.

    <p>False</p> Signup and view all the answers

    What are the three types of bacterial cells after transformation?

    <p>Cells without plasmids, cells with plasmids without foreign DNA, and cells with plasmids with foreign DNA.</p> Signup and view all the answers

    The enzyme β-galactosidase is active only in E. coli that have plasmids without any cloned DNA within the __________.

    <p>polylinker</p> Signup and view all the answers

    Match the cloning method to its description:

    <p>Calcium phosphate = Uses calcium to co-precipitate and introduce DNA into cells Electroporation = Applies high voltage to create transient holes in the cell membrane</p> Signup and view all the answers

    Why is directional cloning particularly important in expression systems?

    <p>It ensures proper orientation of the DNA insert for expression.</p> Signup and view all the answers

    The lac Z gene is responsible for the production of an inactive form of β-galactosidase.

    <p>True</p> Signup and view all the answers

    What role does the lac Z' gene play in recombinant DNA technology?

    <p>It encodes the α-peptide necessary for the active form of β-galactosidase.</p> Signup and view all the answers

    Bacterial transformation involves making the bacterial membrane __________ to allow plasmid entry.

    <p>transiently porous</p> Signup and view all the answers

    Which method is NOT commonly used for bacterial transformation?

    <p>Microinjection</p> Signup and view all the answers

    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.

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