Recombinant DNA-Based Molecular Techniques: DNA Cloning (Part I)
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Questions and Answers

What is the main purpose of DNA cloning?

  • Creating palindromic sequences
  • Producing restriction endonucleases
  • Generating recombinant DNA (correct)
  • Amplifying RNA segments

    • What is the role of a vector in DNA cloning?

  • Breaks the phosphodiester bond
  • Encodes a protein or non-coding RNA
  • Generates sticky ends
  • Serves as a carrier for the gene of interest (correct)

    • How do restriction endonucleases function in DNA cloning?

  • Create recombinant DNA
  • Generate blunt-ended fragments
  • Recognize and cut DNA at specific sequences (correct)
  • Form sticky or cohesive ends

    • What defines palindromic sequences in the context of DNA cloning?

    <p>Sequences that read the same forwards and backwards</p> Signup and view all the answers

    How are blunt-ended fragments generated by restriction endonucleases?

    <p>By cutting at the same position on both strands</p> Signup and view all the answers

    What distinguishes staggered (off-center) cuts by restriction endonucleases?

    <p>They cut the two DNA strands at different positions</p> Signup and view all the answers

    What is the purpose of a DNA ligase in the process of making recombinant DNA?

    <p>To 'close' the plasmid</p> Signup and view all the answers

    What characteristic of an expression vector distinguishes it from a regular vector?

    <p>It contains promoter and termination sequences</p> Signup and view all the answers

    Why is it challenging to express proteins in bacteria?

    <p>They have internal disulfide bonds</p> Signup and view all the answers

    What solution is proposed to overcome the challenges of protein expression in bacteria?

    <p>Employing eukaryotic systems like yeast</p> Signup and view all the answers

    How can proteins be 'tagged' during the process of creation of protein hybrids?

    <p>Inserting a sequence of amino acids called tags</p> Signup and view all the answers

    Which enzyme is crucial for sealing the DNA fragments after they hybridize in the process of making recombinant DNA?

    <p>DNA ligase</p> Signup and view all the answers

    Which protein tag is detected using an antibody specific to a peptide sequence?

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

    What is a protein domain based on the provided text?

    <p>A region of the protein's polypeptide chain with a defined three-dimensional structure</p> Signup and view all the answers

    Why is Green Fluorescent Protein (GFP) used according to the text?

    <p>For protein detection purposes</p> Signup and view all the answers

    For which protein tag is purification achieved by binding to nickel or imidazole?

    <p>Poly-His</p> Signup and view all the answers

    Which method involves separating proteins based on size in gel wells?

    <p>Gel electrophoresis</p> Signup and view all the answers

    Which protein tag is used for protein purification purposes?

    <p>GST-tag</p> Signup and view all the answers

    Which protein tag is associated with a ~220 amino acid protein and can be detected by fluorescence?

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

    What makes a protein domain distinct according to the text?

    <p>It has a defined three-dimensional structure and folds independently</p> Signup and view all the answers

    What is the purpose of the GST-tag in the context provided?

    <p>For purification of GST-tagged proteins</p> Signup and view all the answers

    Which amino acid sequence is specific to the V5 protein tag?

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

    What is the purpose of adding six histidines to a protein?

    <p>Purification using nickel or imidazole</p> Signup and view all the answers

    What is the role of DNA ligase in the context of cloning?

    <p>Joins DNA ends by forming phosphodiester bonds</p> Signup and view all the answers

    What is the purpose of cloning in genetics?

    <p>To make several copies of one thing</p> Signup and view all the answers

    What is recombinant DNA technology?

    <p>Manipulating DNA for various purposes</p> Signup and view all the answers

    Why are bacterial plasmids considered excellent vectors for cloning?

    <p>They replicate independently of the main bacterial genome</p> Signup and view all the answers

    Which feature must plasmid cloning vectors have?

    <p>Presence of an antibiotic resistance gene</p> Signup and view all the answers

    What is the main purpose of using restriction endonucleases in DNA cloning?

    <p>Generate sticky or blunt ends on DNA fragments</p> Signup and view all the answers

    Which of the following accurately describes a palindromic sequence in the context of DNA cloning?

    <p>A sequence that reads the same from left to right as it does from right to left</p> Signup and view all the answers

    In DNA cloning, what is the significance of generating sticky or cohesive ends on DNA fragments?

    <p>Allows for easier insertion into recipient cells</p> Signup and view all the answers

    What characterizes a blunt-ended fragment generated by restriction endonucleases?

    <p>Aligns perfectly with complementary sequences in recipient cells</p> Signup and view all the answers

    What role do vectors play in the process of DNA cloning?

    <p>Carry and replicate inserted genes in host cells</p> Signup and view all the answers

    Why are restriction endonucleases crucial in creating recombinant DNA?

    <p>To generate DNA fragments with specific ends for insertion</p> Signup and view all the answers

    Which enzymatic activity is characteristic of restriction endonucleases?

    <p>Recognize and cut specific sequences in DNA</p> Signup and view all the answers

    What is the purpose of adding six histidines to a protein?

    <p>Enabling purification using nickel-bound beads</p> Signup and view all the answers

    Which protein tag is associated with the amino acid sequence DYKDDDDK?

    <p>FLAG tag</p> Signup and view all the answers

    What method is used to separate proteins according to size in gel electrophoresis?

    <p>Gel electrophoresis (SDS-PAGE)</p> Signup and view all the answers

    Which protein tag allows for purification by binding to glutathione?

    <p>GST tag</p> Signup and view all the answers

    Which amino acid sequence is specific to the V5 protein tag?

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

    What is the detection method for Green Fluorescent Protein (GFP)?

    <p>Fluorescence detection</p> Signup and view all the answers

    What is the purpose of the ShineDalgarno (SD) sequences in expression vectors?

    <p>Promote transcription initiation</p> Signup and view all the answers

    In DNA cloning, what is the function of a poly-T primer?

    <p>Selecting for human mRNA</p> Signup and view all the answers

    Why are eukaryotic systems like yeast preferred over bacteria for protein expression?

    <p>To enable post-translational modifications</p> Signup and view all the answers

    What is the primary role of a gene clone in the creation of protein hybrids?

    <p>Creating tagged proteins</p> Signup and view all the answers

    Which characteristic of restriction endonucleases distinguishes the 'many types of RNA' challenge?

    <p>Palindromic sequences</p> Signup and view all the answers

    What is the impact of the lack of glycosylation on protein expression in bacteria?

    <p>Prevents post-translational modifications</p> Signup and view all the answers

    How does the addition of tags affect protein purification efficiency?

    <p>Improves target protein yield</p> Signup and view all the answers

    What is the function of DNA ligase in DNA cloning?

    <p>Joining DNA ends by forming phosphodiester bonds</p> Signup and view all the answers

    Why are plasmids considered suitable vectors for cloning?

    <p>They possess antibiotic resistance genes</p> Signup and view all the answers

    What is the purpose of an origin of replication in plasmid cloning vectors?

    <p>To ensure independent replication of the plasmid</p> Signup and view all the answers

    How can a DNA fragment be inserted into a plasmid vector?

    <p>By ligating the fragment into the vector</p> Signup and view all the answers

    What is the purpose of a selectable gene in a plasmid cloning vector?

    <p>To identify cells containing the vector</p> Signup and view all the answers

    In cloning, what is the role of recombinant DNA technology?

    <p>Inserting DNA fragments into vectors</p> Signup and view all the answers

    What is a protein domain characterized by?

    <p>Self-stabilization and independent folding</p> Signup and view all the answers

    Which of the following is NOT a characteristic of a protein domain?

    <p>Dependence on the rest of the protein</p> Signup and view all the answers

    What is the role of Green Fluorescent Protein (GFP) in proteins?

    <p>To allow protein detection</p> Signup and view all the answers

    How do GFP-tagged proteins differ from GST-tagged proteins?

    <p>GFP allows for purification purposes</p> Signup and view all the answers

    Which technique involves separating proteins based on size in gel wells?

    <p>Gel electrophoresis</p> Signup and view all the answers

    Why is it essential for a protein domain to fold independently from the rest of the protein?

    <p>To maintain its compact structure</p> Signup and view all the answers

    What distinguishes Green Fluorescent Protein (GFP) from Glutathione S transferase (GST) in their usage?

    <p>Purification vs. gene detection</p> Signup and view all the answers

    What type of structure does a protein domain have?

    <p>$\alpha$-helix, $\beta$-sheet, or both</p> Signup and view all the answers

    What is the purpose of a luciferase reporter assay in molecular biology?

    <p>To study the activity of a gene under certain conditions</p> Signup and view all the answers

    In the Co-immunoprecipitation technique, what is targeted by the antibody molecules conjugated to special beads?

    <p>The protein of interest</p> Signup and view all the answers

    What is the primary role of a DNA library in molecular biology?

    <p>To store DNA fragments for analysis</p> Signup and view all the answers

    Which technique involves transfected cells and measures the expression level of luciferase instead of the original gene?

    <p>Luciferase reporter assay</p> Signup and view all the answers

    What is the main target in Co-immunoprecipitation experiments using antibody-conjugated beads?

    <p>Protein of interest</p> Signup and view all the answers

    Why would a scientist use a luciferase reporter assay instead of directly measuring the original gene's expression level?

    <p>To avoid altering the original gene</p> Signup and view all the answers

    What is the purpose of the LacZ gene in the yeast two-hybrid system?

    <p>To generate a blue product in the presence of X-gal</p> Signup and view all the answers

    Why are both DNA-binding domain (BD) and activation domain (AD) required to be close to each other in the yeast two-hybrid system?

    <p>To activate transcription</p> Signup and view all the answers

    How is the production of recombinant proteins achieved in the cloning of hybrid proteins?

    <p>By transferring recombinant plasmids into yeast cells</p> Signup and view all the answers

    What is the purpose of transferring recombinant plasmids into yeast cells in the cloning of hybrid proteins?

    <p>To identify unknown interacting proteins</p> Signup and view all the answers

    Why are yeast cells grown in the presence of X-gal during the yeast two-hybrid system?

    <p>To identify interacting proteins based on color change</p> Signup and view all the answers

    What distinguishes the function of DNA-binding domain (BD) and activation domain (AD) in the yeast two-hybrid system?

    <p>BD controls DNA binding, while AD enhances transcription activation.</p> Signup and view all the answers

    What is the main purpose of a luciferase reporter assay in molecular biology?

    <p>Study the activity of a gene by measuring luciferase expression</p> Signup and view all the answers

    In a luciferase reporter assay, what is placed upstream of the reporter gene in the plasmid?

    <p>Regulatory region of the gene</p> Signup and view all the answers

    What is the specific function of Co-immunoprecipitation in molecular techniques?

    <p>Targeting proteins bound to beads</p> Signup and view all the answers

    In Co-immunoprecipitation, what are antibody molecules conjugated to for targeting proteins?

    <p>Special beads</p> Signup and view all the answers

    What distinguishes Firefly luciferase in a luciferase reporter assay?

    <p>Role as a reporter gene</p> Signup and view all the answers

    What is the primary purpose of a DNA library?

    <p>Create bacterial clones with specific DNA pieces</p> Signup and view all the answers

    What is the role of the promoter in a luciferase reporter assay using recombinant DNA techniques?

    <p>'Switch on' luciferase expression</p> Signup and view all the answers

    How does Co-immunoprecipitation work in identifying proteins of interest?

    <p>'Switch on' only the protein of interest and its bound proteins</p> Signup and view all the answers

    What is the role of the activation domain (AD) in the yeast two-hybrid system?

    <p>Responsible for the activation of transcription</p> Signup and view all the answers

    Why are both DNA-binding domain (BD) and activation domain (AD) required to be close to each other in the yeast two-hybrid system?

    <p>To facilitate interactions between known and unknown proteins</p> Signup and view all the answers

    What distinguishes the UAS in yeast cells?

    <p>Is controlled by a transcription factor</p> Signup and view all the answers

    What is the significance of growing yeast cells in the presence of X-gal during the yeast two-hybrid system?

    <p>Produces a blue product upon cleavage</p> Signup and view all the answers

    In the context of cloning hybrid proteins, what is cloned separately along with the unknown gene Y?

    <p>The activation domain (AD)</p> Signup and view all the answers

    What is the primary purpose of using the LacZ gene in the yeast two-hybrid system?

    <p>To generate a blue product for identifying interactions</p> Signup and view all the answers

    What is the main function of Cas9 in the CRISPR/Cas9 system?

    <p>Creating single or double strand breaks in DNA</p> Signup and view all the answers

    How is the Cas9 nuclease directed to its target sequence in the CRISPR/Cas9 system?

    <p>By a short RNA fragment known as a guide RNA (gRNA)</p> Signup and view all the answers

    What happens when a phage infects a bacterial cell in relation to the CRISPR/Cas9 system?

    <p>The bacterial cell degrades the phage DNA and integrates fragments into the CRISPR cluster</p> Signup and view all the answers

    How can a gene be studied using CRISPR/Cas9 gene editing technology?

    <p>By directly altering the gene's function using Cas9</p> Signup and view all the answers

    What is the specific role of the guide RNA (gRNA) in the CRISPR/Cas9 system?

    <p>Directing Cas9 to its target sequence in DNA</p> Signup and view all the answers

    What is the outcome of introducing both the gRNA and Cas9 gene into human cells?

    <p>Study of gene function by mutating it</p> Signup and view all the answers

    How does CRISPR/Cas9 system protect bacterial cells from phages?

    <p>By degrading the phage DNA and integrating a fragment into the CRISPR cluster</p> Signup and view all the answers

    What guides the Cas9 nuclease to its target sequence in the CRISPR/Cas9 system?

    <p>Guide RNA (gRNA)</p> Signup and view all the answers

    What allows for the function of a gene to be studied through gene editing mechanisms?

    <p>Mutating the gene to observe effects</p> Signup and view all the answers

    How are both gRNA and Cas9 gene introduced into human cells in gene editing?

    <p>As separate genes cloned into plasmid vectors</p> Signup and view all the answers

    What specifically occurs when a phage infects a bacterial cell after integration into the CRISPR/Cas9 system?

    <p>The phage DNA is transcribed into guide RNA (gRNA)</p> Signup and view all the answers

    What is the role of Cas9 nuclease in gene editing using CRISPR technology?

    <p>Guiding to its target DNA sequence for cutting</p> Signup and view all the answers

    How does gene editing with CRISPR technology enable replacing a mutated gene with a normal one?

    <p>Through DNA repair mechanisms using introduced DNA</p> Signup and view all the answers

    'What role does gRNA play in guiding Cas9 nuclease to its target?' - What would be a correct answer?

    <p>'Directing Cas9 to cut specific DNA sequences'</p> Signup and view all the answers

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