DNA Engineering and Cloning Quiz
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Questions and Answers

Which method involves the use of a high voltage pulse to introduce recombinant DNA into a host cell?

  • Biolistics
  • Electroporation (correct)
  • Conjugation
  • Heat shock method

    • What is the primary purpose of selectable markers in cloning vectors?

  • To allow identification of transformed cells (correct)
  • To enhance gene expression
  • To promote faster DNA replication
  • To increase the size of the vector

    • What distinguishes a screenable marker from a selectable marker?

  • Selectable markers are always antibiotic resistance genes
  • Screenable markers kill non-transformed cells
  • Screenable markers require specific environmental conditions for expression
  • Screenable markers produce a visible characteristic without harming cells (correct)

    • Which of the following is NOT a method of transformation?

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

    What happens to bacterial cells that contain a disrupted LacZ gene when grown on X-gal medium?

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

    The blue-white selection technology is used to distinguish between which two types of colonies?

    <p>Bacteria harboring vectors with insert and without insert</p> Signup and view all the answers

    How do selectable markers function during the transformation process?

    <p>By allowing for growth in the presence of specific antibiotics</p> Signup and view all the answers

    Contamination with undigested vector during cloning can lead to what issue?

    <p>Failure to acquire the desired DNA fragment</p> Signup and view all the answers

    What color do bacteria harboring the pUC vector with insert DNA appear when grown in the presence of X-Gal?

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

    What is the product formed when β-galactosidase cleaves X-Gal?

    <p>Galactose and an insoluble blue compound</p> Signup and view all the answers

    What is a common method to ensure that host cells contain the correct recombinant DNA?

    <p>Gel electrophoresis to analyze DNA size</p> Signup and view all the answers

    Which type of selection does the pUC vector use to differentiate between colonies?

    <p>Color-based selection</p> Signup and view all the answers

    How is DNA separated to determine the size of fragments during gel electrophoresis?

    <p>By charge and size</p> Signup and view all the answers

    What is the role of IPTG in the context of the pUC vector?

    <p>It induces the expression of β-galactosidase</p> Signup and view all the answers

    Which statement about the pUC vector is false?

    <p>The recombinant DNA can only be identified by color change.</p> Signup and view all the answers

    What type of gel is recommended for separating DNA fragments?

    <p>Agarose gel</p> Signup and view all the answers

    What is a key function of DNA ligases during the cloning process?

    <p>To join DNA ends at strand breaks</p> Signup and view all the answers

    Which of the following statements is true regarding the use of a single restriction enzyme (RE)?

    <p>It may result in self-ligation of the vector.</p> Signup and view all the answers

    What is a limitation of using an RE that is present in the middle of the gene to be cloned?

    <p>It can prevent the amplification of the entire gene.</p> Signup and view all the answers

    In the context of PCR, why is it important to create RE sites before ligation?

    <p>To ensure compatibility with the vector being used.</p> Signup and view all the answers

    What is the function of sticky ends produced during cloning?

    <p>To promote specific interactions between DNA fragments.</p> Signup and view all the answers

    Why might a researcher choose to use a plasmid vector for cloning instead of chromosomal DNA?

    <p>Plasmid vectors are smaller and more easily manipulated.</p> Signup and view all the answers

    Which factor is NOT a consideration when selecting RE sites on the DNA to be cloned?

    <p>The affinity of the RE for the target sequence</p> Signup and view all the answers

    What is the expected result of using an RE that leads to concatemer formation?

    <p>A long continuous DNA strand may form.</p> Signup and view all the answers

    Study Notes

    DNA Engineering and Cloning

    • Lecture Objectives: Learn about restriction enzymes, cloning DNA fragments in plasmids, transforming recombinant plasmids into bacterial cells, and different bacterial and eukaryotic vectors.
    • Text Section: 7.1.20.1.
    • Additional Reading: Material available on Canvas.

    Cloning DNA/Gene

    • Clone: An identical copy of a single mother cell.
    • DNA/Gene Cloning Process:
      • Separates a specific gene or DNA fragment from a larger DNA molecule (e.g., chromosome).
      • Inserts the specific fragment into a vector (plasmid or carrier DNA).
      • Introduces the "construct" into a host cell.
      • Replicates the DNA in the host cell.

    Restriction Endonucleases (REs)

    • RE Function: Recognize and cut DNA at specific nucleotide sequences.
    • RE Types:
      • Type I: Cuts DNA at a random site far from the recognition site (hundreds of bases). Recognition site is asymmetrical, with 3-4 nucleotides in one section and 4-5 nucleotides in another.
      • Type II: Cuts DNA within the recognition site. Recognition site is palindromic and 4-8 nucleotides long. Commonly used in DNA work.
      • Type III: Cuts DNA about 20-30 base pairs after the recognition site. Recognizes two inversely oriented non-palindromic sequences.
    • Sticky Ends: Produced by some REs and can base-pair with each other or with complementary sticky ends.
    • Blunt Ends: Produced by other REs.
    • Base Cutters: Most frequently used REs in molecular biology are 6 base cutters.

    Frequency of Type II RE Cutting

    • Cutting Frequency Calculation: Y = 4n, where Y = expected number of cuts and n = number of bases in the recognition site.
      • 6 bp cutter: Cuts once every 46 (4,096) base pairs, on average.
      • 4 bp cutter: Cuts once every 44 (256) base pairs, on average.

    Cloning DNA/Gene Steps

    • 1. Select a vector (Fig 7-4).
    • 2. Select appropriate restriction enzymes (REs) for the target gene and vector.
    • 3. Digest the vector and target gene with the appropriate REs.
    • 4. Ligate the target gene to the vector.
    • 5. Transform the recombinant DNA into bacteria.
    • 6. Select transformed bacteria.
    • 7. Ensure host cells carry the recombinant DNA.

    Cloning Vectors

    • Purpose: DNA "vehicles" capable of self-replication, used for cloning, maintaining, and delivering DNA to biological systems.
    • Types: Modified versions of naturally occurring plasmids (small DNA molecules in bacteria) or small viral DNA.
    • Key Features:
      • Origin of replication (ori)
      • Selectable marker (e.g., TetR or AmpR)
      • Screenable marker (e.g., GFP and LacZ)
      • Multiple Cloning Site (MCS)
      • Relatively small size

    Vector Classes

    • i) Housekeeping: pBR322, pUC18/19 etc.
    • ii) Expression: pET and pGEX vectors.
    • iii) Delivery (Transformation): pCAMBIA (for plant transformation).

    pUC19 Vector

    • Ideal for: Gene cloning and maintenance.

    Selecting RE Sites on DNA

    • a) Locating Existing Sites: Software is available online (e.g., http://molbiol-tools.ca/) to find RE sites in a DNA sequence.
    • b) Creating New Sites: PCR can be used (strategy 2).

    Digesting DNA with REs

    • REs cut at specific sequences, creating either sticky or blunt ends.

    Ligation

    • DNA ligases join DNA ends (strand breaks or nicks).
      • The 5' terminus must be phosphorylated.
      • The 3' terminus must have a free OH group.

    Concatemer Concerns

    • If only one RE is used, the target DNA may form a concatemer. This can create issues.

    Transforming DNA into a Host Organism

    • Methods: Heat shock, electroporation, conjugation, biolistics (gene gun).

    Identifying Transformed Host Cells

    • Selectable Markers: Genes (e.g., antibiotic resistance) that allow growth of cells under specific conditions (transformation).
      • Common Antibiotics: Ampicillin, kanamycin, and tetracycline.
    • Screenable Markers: To visually identify cells and easier to select transformed colonies.
      • Example: LacZ gene.

    Creating Sticky Ends via PCR

    • Designed PCR primers
    • Amplify the target gene in two separate reactions
    • Purify/clean PCR products
    • Mix products equally
    • Denature and re-nature to generate desired sticky ends

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    Related Documents

    Lecture 7 (Cloning DNA) Slides (PDF)

    Description

    Test your knowledge on DNA engineering, including the processes of gene cloning and the use of restriction enzymes. This quiz covers key topics from the lecture, focusing on the mechanisms of cloning and the role of various vectors in biotechnology.

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