Podcast
Questions and Answers
What is the primary function of alkaline phosphatase in DNA modification?
What is the primary function of alkaline phosphatase in DNA modification?
- Attaches 5′-phosphate groups
- Cuts DNA molecules at specific positions
- Adds deoxyribonucleotides to the 3′ terminus
- Removes 5′-phosphate groups (correct)
What is the role of terminal deoxynucleotidyl transferase in DNA modifications?
What is the role of terminal deoxynucleotidyl transferase in DNA modifications?
- Removes nucleotides from DNA strands
- Phosphorylates the DNA molecule
- Cuts DNA at specific sequences
- Adds deoxyribonucleotides to the 3′ terminus (correct)
Why is precise cutting of DNA essential in gene cloning?
Why is precise cutting of DNA essential in gene cloning?
- To increase the overall length of the DNA
- To ensure random fragmentation of DNA
- To enhance the stability of the DNA molecule
- To allow for the insertion of new DNA at specific locations (correct)
What does a restriction endonuclease specifically do?
What does a restriction endonuclease specifically do?
What will happen if a DNA vector is cut at multiple locations?
What will happen if a DNA vector is cut at multiple locations?
What must happen to large DNA molecules before cloning a specific gene?
What must happen to large DNA molecules before cloning a specific gene?
What is the function of polynucleotide kinase in DNA modification?
What is the function of polynucleotide kinase in DNA modification?
Which of the following is NOT a characteristic of restriction endonucleases?
Which of the following is NOT a characteristic of restriction endonucleases?
What primary action differentiates exonucleases from endonucleases?
What primary action differentiates exonucleases from endonucleases?
Which statement about Bal31 exonuclease is accurate?
Which statement about Bal31 exonuclease is accurate?
How does exonuclease III differ from Bal31?
How does exonuclease III differ from Bal31?
Which of the following accurately describes S1 endonuclease?
Which of the following accurately describes S1 endonuclease?
What is the unique characteristic of DNase I?
What is the unique characteristic of DNase I?
What distinguishes restriction endonucleases from other endonucleases?
What distinguishes restriction endonucleases from other endonucleases?
Why would prolonged action of DNase I result in a mixture of mononucleotides and short oligonucleotides?
Why would prolonged action of DNase I result in a mixture of mononucleotides and short oligonucleotides?
What is the main effect of Bal31 on double-stranded DNA over time?
What is the main effect of Bal31 on double-stranded DNA over time?
What type of enzyme is responsible for cutting, shortening, or degrading nucleic acid molecules?
What type of enzyme is responsible for cutting, shortening, or degrading nucleic acid molecules?
Which type of enzyme joins nucleic acid molecules together?
Which type of enzyme joins nucleic acid molecules together?
What is the primary role of polymerases in DNA manipulation?
What is the primary role of polymerases in DNA manipulation?
What is a common activity of some polymerases aside from making new DNA molecules?
What is a common activity of some polymerases aside from making new DNA molecules?
Which enzymes are known to act on RNA?
Which enzymes are known to act on RNA?
What is the function of modifying enzymes in DNA operations?
What is the function of modifying enzymes in DNA operations?
Which of the following is NOT a broad class of DNA manipulative enzymes?
Which of the following is NOT a broad class of DNA manipulative enzymes?
What cellular process involves the breakdown of unwanted or foreign DNA?
What cellular process involves the breakdown of unwanted or foreign DNA?
What characteristic do sticky ends have that allows them to recombine DNA molecules?
What characteristic do sticky ends have that allows them to recombine DNA molecules?
Which of the following restriction endonucleases produces the same sticky ends?
Which of the following restriction endonucleases produces the same sticky ends?
How often should a tetranucleotide sequence like GATC theoretically occur in a DNA strand?
How often should a tetranucleotide sequence like GATC theoretically occur in a DNA strand?
What is a common reason for the discrepancy between theoretical and actual recognition sites for restriction endonucleases in DNA?
What is a common reason for the discrepancy between theoretical and actual recognition sites for restriction endonucleases in DNA?
What does it mean if a restriction site is not evenly spaced along the DNA molecule?
What does it mean if a restriction site is not evenly spaced along the DNA molecule?
How many recognition sequences would you expect from a hexanucleotide in a 49 kb DNA molecule?
How many recognition sequences would you expect from a hexanucleotide in a 49 kb DNA molecule?
Which of the following statements about blunt ends is correct?
Which of the following statements about blunt ends is correct?
What is the effect of a lower than expected GC content in a DNA strand?
What is the effect of a lower than expected GC content in a DNA strand?
What are the benefits of type II restriction enzymes over type I and III systems?
What are the benefits of type II restriction enzymes over type I and III systems?
How does the restriction activity of type IIs systems differ compared to type II systems?
How does the restriction activity of type IIs systems differ compared to type II systems?
What is a key characteristic of homing endonucleases?
What is a key characteristic of homing endonucleases?
What information does the naming system for restriction endonucleases provide?
What information does the naming system for restriction endonucleases provide?
Why may the classification of R-M systems require modification?
Why may the classification of R-M systems require modification?
In what way do type II restriction enzymes usually cut DNA?
In what way do type II restriction enzymes usually cut DNA?
What generally limits the usefulness of type IIs systems?
What generally limits the usefulness of type IIs systems?
Which feature is NOT a characteristic of type II restriction enzymes?
Which feature is NOT a characteristic of type II restriction enzymes?
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Study Notes
Nucleases
- Cut, shorten, or degrade nucleic acid molecules
- Break phosphodiester bonds linking nucleotides in a DNA strand
- Two types:
- Exonucleases: Remove nucleotides one at a time from the end of a DNA molecule
- Bal31: Removes nucleotides from both strands of a double-stranded DNA molecule
- Exonuclease III: Removes nucleotides only from the 3’ terminus
- Endonucleases: Break internal phosphodiester bonds within a DNA molecule
- S1 endonuclease: Cleaves only single strands of DNA
- Deoxyribonuclease I (DNase I): Cuts both single and double-stranded molecules
- Non-specific, attacks any internal phosphodiester bond
- Results in a mixture of mononucleotides and very short oligonucleotides
- Restriction endonucleases (RE): Cleave double-stranded DNA only at specific recognition sites
- Exonucleases: Remove nucleotides one at a time from the end of a DNA molecule
DNA Manipulative Enzymes
- Classified into four classes depending on the type of reaction:
- Nucleases
- Ligases
- Polymerases
- Modifying enzymes
- Many enzymes exhibit multiple activities spanning two or more classes
- Many polymerases combine their ability to make new DNA with DNA degradative (nuclease) activity
- Enzymes that act on RNA are known, but the focus is on those that act on DNA
DNA Ligases
- Join nucleic acid molecules together
- Form phosphodiester bonds between the 5’ phosphate group at the end of one DNA strand and the 3’ hydroxyl group at the end of another strand
DNA Polymerases
- Make copies of DNA molecules
- Need a template strand to direct the synthesis of a new complementary strand
- Typically create phosphodiester bonds between nucleotides
DNA Modifying Enzymes
- Remove or add chemical groups
- Examples:
- Alkaline phosphatase: Removes 5' phosphate groups
- Polynucleotide kinase: Attaches 5' phosphate groups
- Terminal deoxynucleotidyl transferase: Adds deoxyribonucleotides to the 3' termini of DNA molecules
Restriction Endonucleases (RE)
- Cut DNA at specific recognition sites
- Highly specific and reproducible, essential for gene cloning
- Classified into types I, II, and III
- Type II enzymes:
- Recognize a defined, usually symmetrical, sequence and cut within it
- Many produce staggered cuts, creating sticky ends
- Do not require cofactors, easier to use
- Naming convention:
- First letter of the genus name
- First two letters of the specific epithet (E.g., EcoRI from Escherichia coli)
- Sticky ends allow fragments to be joined together
- The frequency of recognition sequences in a DNA molecule can be calculated mathematically
- Tetranucleotide sequence occurs once every 256 nucleotides
- Hexanucleotide sequence occurs once every 4096 nucleotides
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