Use of restriction enzymes and ligases

• How do Type I restriction enzymes differ from Type II enzymes?

  Type I cuts far from recognition sites
• What are the characteristics of Type I and Type II restriction enzymes?

  • Type I:
  • Large enzymes
  • Cut DNA far from recognition sites
  • Type II:
  • Smaller enzymes
  • Cut DNA at specific palindromic sequences
• Why do restriction enzymes cut specific palindromic DNA sequences?

  Because the palindromic sequence allows the enzyme to recognize and bind to the DNA in a symmetric way
• How can restriction enzymes be compared to common tools?

  They are like molecular scissors
• What is the 5' to 3' sequence of the DNA palindrome shown in the image?

  AGTACTCATGA
• How do the two restriction enzymes shown in the image differ in their recognition sequences?

  • Enzyme 1 recognizes the sequence TGCGCA
  • Enzyme 2 recognizes the sequence GAATTC
• Why are Type II restriction enzymes useful in genetic engineering?

  They cut DNA at specific sequences
• What do Type II restriction enzymes cut DNA at?

  Specific palindromic sequences
• If you wanted to cut a DNA sequence into the smallest possible fragments, which type of restriction enzyme would you use?

  Enzyme 1, which has a longer 6 base pair recognition sequence
• What type of sequences do restriction enzymes cut?

  Palindromic sequences
• Why are restriction enzymes important for genetic engineers?

  They allow precise cutting and pasting of DNA
• How do the recognition sequences of the two restriction enzymes shown differ in length?

  Enzyme 1 has a longer recognition sequence (6 base pairs) than Enzyme 2 (4 base pairs)
• What are the two types of DNA ends produced by restriction enzymes?

  • Sticky end overhang
  • Blunt end (no overhang)
• What is the primary function of restriction enzymes?

  To cut DNA at specific sites
• If a DNA sequence has the palindromic sequence GAATTC, which restriction enzyme from the image would cut it?

  Enzyme 2
• What is the primary function of restriction enzymes in bacteria?

  To protect against viral infections
• How do palindromic sequences read on DNA strands?

  They read the same backwards on opposite strands
• What is a DNA palindrome?

  A sequence which, when flipped, is identical to the complementary sequence
• How do the sequences recognized by different enzymes illustrate specificity?

  Each enzyme cuts at unique palindromic sequences
• What are the two main types of restriction enzymes?

  Type I and Type II
• What are the key differences between the sticky end overhang and blunt end produced by the restriction enzymes?

  • Sticky end has a single-stranded overhang, blunt end has no overhang
  • Sticky end allows for easier ligation, blunt end is more difficult to ligate
  • Sticky end provides more specificity in DNA fragment assembly, blunt end is less specific
• What are the characteristics of DNA fragments produced by restriction enzymes?

  • Created by cutting DNA at recognition sites
  • Can have blunt or sticky ends
  • Result from breaking phosphodiester bonds
• What are restriction enzymes also known as?

  Restriction endonucleases
• What is the name of the enzyme that cuts the DNA strands?

  DNA ligase
• What are restriction enzymes used for in DNA digestion?

  They cut DNA at specific recognition sites
• What is the name of the restriction enzyme that recognizes the sequence GAATTC/CTTAAG?

  EcoRI
• What is the name of the biological process shown in the image?

  DNA ligase
• What energy source is required for DNA ligase to function?

  ATP energy
• What analogy is used to describe the function of DNA ligases?

  They act like tape for DNA fragments
• How does DNA ligase seal the gaps between DNA strands?

  • DNA ligase catalyzes the formation of phosphodiester bonds
  • This seals the gaps between Okazaki fragments during DNA replication
  • It joins the 3' hydroxyl end of one DNA strand to the 5' phosphate end of another
• Why is DNA ligase an important enzyme for DNA repair and replication?

  It joins DNA fragments together to maintain the integrity of the DNA molecule
• What type of bonds does DNA ligase form to seal gaps?

  Phosphodiester bonds
• What is the purpose of the single-stranded overhangs left by the restriction enzyme?

  The overhangs allow the DNA fragments to base-pair and stick together
• What is required for the process of DNA ligation?

  ATP
• What are the cofactors required for DNA ligase activity?

  • ATP or NAD+
  • Magnesium ions (Mg2+)
• What is the name of the enzyme shown in the image?

  Restriction enzyme
• What is the structure shown in the image?

  DNA
• What types of ends can DNA fragments have after being cut by restriction enzymes?

  Blunt or sticky ends
• How does the activity of DNA ligase differ in DNA replication versus DNA repair?

  In replication, it joins Okazaki fragments, while in repair it seals breaks in the DNA backbone
• What are sticky ends in the context of DNA digestion?

  Single-stranded DNA overhangs