Biotechnology Overview: Plasmids & Recombinant DNA
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Questions and Answers

What is the main purpose of recombinant DNA technology?

  • To create new plasmids without bacteria
  • To solely produce proteins
  • To enhance or suppress gene expression (correct)
  • To study bacterial growth patterns

    • Type II restriction endonucleases are not suitable for gene cloning.

    False

    What is a plasmid?

    A circular self-replicating DNA molecule separate from bacterial DNA.

    Restriction endonucleases are also known as __________.

    <p>restriction enzymes</p> Signup and view all the answers

    Match the following types of restriction endonucleases with their characteristics:

    <p>Type I = Cleaves DNA at sites other than the recognition site Type II = Widely used for gene cloning Type III = Also cleaves DNA at sites other than the recognition site</p> Signup and view all the answers

    Which of the following is NOT a use of recombinant DNA technology?

    <p>Synthesizing antibiotics</p> Signup and view all the answers

    What is one disadvantage of the single restriction method of inserting foreign DNA into plasmid vectors?

    <p>Difficult to achieve directional cloning</p> Signup and view all the answers

    The polylinker affects the transcription of the lac Z' gene.

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

    What two restriction enzymes are typically used in the double restriction approach mentioned?

    <p>BamHI and EcoRI</p> Signup and view all the answers

    The process of making the bacterial membrane transiently porous to allow plasmid entry is called __________.

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

    Match the following techniques with their descriptions:

    <p>Single restriction = Involves cutting with one enzyme allowing insert in both orientations Double restriction = Uses two enzymes to ensure correct insert orientation Transformation = Process of getting the recombinant plasmid into bacterial cells Directional cloning = Inserts DNA in a specific orientation relative to control sequences</p> Signup and view all the answers

    Which of the following is NOT a source of foreign DNA for cloning?

    <p>PCR-amplified RNA</p> Signup and view all the answers

    The plasmid pBR322 is an example of a high copy number plasmid.

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

    What does the lowercase 'p' in plasmid vectors stand for?

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

    An ideal bacterial plasmid vector must contain an origin of _______ to allow independent replication.

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

    Match the plasmid with its description:

    <p>pBR322 = Has antibiotic resistance genes pUC18 = High copy number plasmid pUC19 = Derivative of pBR322</p> Signup and view all the answers

    What is a key feature of a multiple cloning site (polylinker) in a plasmid vector?

    <p>Contains unique restriction endonuclease cleavage sites.</p> Signup and view all the answers

    Stringent control of plasmid replication allows for many copies of the plasmid in each bacterial cell.

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

    What type of genetic selection is used to distinguish transformed host cells from untransformed ones?

    <p>Antibiotic resistance or blue-white screening</p> Signup and view all the answers

    Modern cloning vectors are often built from _______ plasmid vectors.

    <p>pUC19</p> Signup and view all the answers

    What feature is crucial for the extraction of plasmids from host cells?

    <p>Efficient and simple extraction method</p> Signup and view all the answers

    What is the function of BamHI in recombinant DNA technology?

    <p>To cut DNA at specific sequences</p> Signup and view all the answers

    T4 DNA ligase is used to covalently link DNA fragments together.

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

    What are the complementary sticky ends generated by BamHI in the provided DNA sequences?

    <p>5’--G G-A-T-C-C--3’ and 3’--C-C-T-A-G G--5’</p> Signup and view all the answers

    Terminal deoxynucleotidyl transferase adds deoxynucleotides to the ______ ends of DNA molecules.

    <p>3′OH</p> Signup and view all the answers

    Match the following enzymes with their functions:

    <p>BamHI = Cuts DNA at specific sequences T4 DNA ligase = Links DNA fragments together DNA polymerase III = Synthesizes DNA from a template Terminal transferase = Adds nucleotides to 3' OH ends of DNA</p> Signup and view all the answers

    What is a key characteristic of the DNA fragments generated by BamHI?

    <p>They have sticky or cohesive ends.</p> Signup and view all the answers

    DNA polymerase III and terminal transferase have the same requirements for DNA synthesis.

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

    What type of ends do the fragments have after cleavage with BamHI?

    <p>Sticky ends</p> Signup and view all the answers

    Study Notes

    Plasmids

    • Circular DNA molecule, separate from bacterial DNA
    • Replicates independently
    • Can be used to introduce foreign DNA into bacteria

    Recombinant DNA Technology

    • Used to study gene arrangement, expression and regulation
    • Modifies gene expression to enhance or suppress products
    • Creates artificial copies of DNA segments
    • Transfers genes between organisms, creating transgenic organisms with altered characteristics
    • Creates transgenic organisms used for commercial purposes

    Restriction Endonucleases (REs)

    • Bacterial enzymes that cut DNA at specific recognition sites
    • Type II REs are used in gene cloning because they cleave DNA at specific sites
    • Important for mapping and reconstructing DNA in vitro

    Terminal Deoxynucleotidyl Transferase (Terminal Transferase)

    • Enzyme that adds deoxynucleotides to the 3' end of DNA molecules
    • Does not have proofreading ability
    • Distinct from DNA polymerase III, which has proofreading ability

    Foreign DNA Cloning Sources

    • Chromosomal DNA
    • mRNA converted to cDNA
    • PCR-amplified DNA

    Cloning and Expression Vectors

    • Carry foreign DNA into bacterial cells
    • Used in gene cloning and protein production
    • Plasmids are named with a system of uppercase letters and numbers, "p" stands for plasmid
    • pBR322, pUC18, p UC19 are examples of commonly used plasmids
    • High copy number plasmids replicate more than 5oo copies per bacterial host cell, occur in plasmids with relaxed replication control
    • Stringent replication is coupled to host chromosome, results in lower copy numbers

    Essential features of a bacterial plasmid vector

    • Origin of replication (ori) for independent replication
    • Selection marker/s for identifying transformed cells:
      • Antibiotic resistance
      • Blue-white screening
    • Multiple cloning site (MCS) containing restriction endonuclease cleavage sites for insertion of foreign DNA
    • Easy extraction from host cells

    Inserting Foreign DNA into Plasmid Vectors

    • Single restriction approach:
      • Can lead to ligation of the restricted plasmid
      • Foreign DNA can be inserted in both orientations
      • Multiple fragments can insert into the excised plasmid
    • Double restriction approach:
      • Uses two restriction enzymes to cleave the plasmid and foreign DNA components
      • Reduces problems associated with single restriction
      • Directional cloning, ensures foreign DNA is inserted in the correct orientation with respect to transcriptional and translational control sequences

    Bacterial Transformation

    • Process of introducing recombinant plasmids into bacterial cells
    • Makes bacterial membrane transiently porous for plasmid uptake

    Cosmids:

    • Plasmids with a cos site (cohesive end site)
    • Can be packaged into phage heads due to the cos site
    • Can carry inserts of 33-47 kbp
    • Combine advantages of plasmids and phage vectors

    Bacterial Artificial Chromosomes (BACs):

    • Based on the fertility plasmid (F plasmid)
    • Designed for cloning large DNA sequences
    • Contain partition genes for even distribution of plasmids during bacterial cell division
    • Accommodate inserts of up to 300 kbp
    • Used in chromosome mapping

    Expression Vectors:

    • Designed for protein expression of target genes
    • Often plasmid-based
    • Utilize T7 RNA polymerase gene expression system for efficient production of proteins
    • Contain transcriptional and translational control sequences for gene expression
    • Transform E.coli host cells with a T7 RNA polymerase gene integrated into its chromosome

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    Description

    Explore the fundamental concepts of plasmids, recombinant DNA technology, and the role of restriction endonucleases. This quiz covers how these elements work together to modify gene expression and create transgenic organisms for various applications. Test your understanding of key enzymes and their functions in molecular biology.

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