L51: Techniques 2- Cloning

• What are the two types of cytoskeletal filaments mentioned?

  • Tubulin microtubules
  • Actin filaments
• What can DNA do in genetic engineering?

  DNA can be cut and pasted together again
• What is the purpose of restriction enzymes?

  They cleave phage DNA to protect host DNA
• How does the host DNA avoid being cleaved by restriction enzymes?

  It is modified by methylation
• In which organisms are restriction enzymes found?

  Bacteria and archaea
• What are the four major classes of restriction enzymes?

  1. Type I
  2. Type II
  3. Type III
  4. Type IV
• What is a characteristic of Type II restriction enzymes?

  They cleave within their recognition site
• What do Type II restriction enzymes typically require?

  Magnesium
• What is a restriction site?

  A specific sequence recognized by restriction enzymes
• What is the typical length of sequences recognized by Type II restriction enzymes?

  4, 6, or 8 bp
• What is a palindromic sequence in DNA?

  A sequence that reads the same in both directions
• What is the cleavage site for BamHI?

  5'-GGATCC-3'
• What is the cleavage site for EcoRI?

  5'-GAATTC-3'
• What is the cleavage site for HaeIII?

  5'-GGCC-3'
• What is the cleavage site for HhaI?

  5'-GCGC-3'
• What is the cleavage site for XhoI?

  5'-CTCGAG-3'
• What is the cleavage site for NotI?

  5'-GCGGCCGC-3'
• What are the features of cloning vectors?

  • Origin of replication (ori)
  • Selectable marker (e.g., antibiotic resistance)
  • Disruptable marker (e.g., lacZ')
  • No conjugation ability
• What is the purpose of a selectable marker in cloning vectors?

  To allow growth of plasmid-containing bacteria
• What is the role of ligases in DNA manipulation?

  To join DNA fragments by repairing backbones
• What are the features of pUC plasmids?

  1. Small size, multi-copy
  2. Origin of replication (ori)
  3. Selectable marker (bla)
  4. Multiple cloning site (MCS)
  5. Disruptable marker (lacZ')
• Why might you want to clone a gene?

  To produce large amounts of the encoded protein
• What is the Central Dogma of molecular biology?

  • DNA makes RNA
  • RNA makes protein
• What does transcription involve?

  Converting DNA into RNA
• What does translation involve?

  Converting RNA into protein
• What is the significance of the 5' and 3' ends in nucleic acids?

  They indicate the directionality of the strand
• What is the role of the lac promoter in gene expression?

  To control the expression of cloned genes
• What is the purpose of using CaCl2 in bacterial transformation?

  To facilitate plasmid uptake by E. coli
• How does electroporation assist in bacterial transformation?

  It creates pores in the bacterial membrane
• What is the process of color selection in cloning?

  • Blue colonies indicate no cleavage
  • White colonies indicate successful cloning
• What is the role of β-galactosidase in cloning?

  It helps identify recombinant plasmids
• What does the term "recombinant protein" refer to?

  A protein produced from cloned DNA
• What is the significance of the Kozak sequence in translation?

  It helps initiate translation in eukaryotes
• What is the function of the Shine-Dalgarno sequence?

  It helps initiate translation in prokaryotes
• What is the role of the start codon (AUG) in translation?

  It signals the beginning of protein synthesis
• What is the role of the stop codons (TGA, UGA) in translation?

  They signal the termination of protein synthesis
• What is the significance of the SD or Kozak sequence in mRNA?

  They assist in the initiation of translation
• What is the relationship between transcription and translation?

  Transcription produces mRNA for translation
• What is the role of the promoter in gene expression?

  It initiates transcription of the gene
• What is the function of the ribosome during translation?

  It assembles amino acids into proteins