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Questions and Answers
An enzyme with a longer recognition sequence, such as ______, cuts at an eight-nucleotide sequence.
An enzyme with a longer recognition sequence, such as ______, cuts at an eight-nucleotide sequence.
NotI
______ would be expected to cut a DNA molecule once every 62 nucleotides.
______ would be expected to cut a DNA molecule once every 62 nucleotides.
NotI
During conventional gel electrophoresis, DNA molecules travel a distance based on their ______.
During conventional gel electrophoresis, DNA molecules travel a distance based on their ______.
size
Poor resolution of DNA is observed above a certain ______ size in gel electrophoresis.
Poor resolution of DNA is observed above a certain ______ size in gel electrophoresis.
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The conventional gel electrophoresis technique differs from ______ field alternation gel electrophoresis (OFAGE).
The conventional gel electrophoresis technique differs from ______ field alternation gel electrophoresis (OFAGE).
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A tetranucleotide sequence occurs once every ______ nucleotides.
A tetranucleotide sequence occurs once every ______ nucleotides.
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The production of ______ ends does not create overhangs.
The production of ______ ends does not create overhangs.
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EcoRI is a restriction enzyme known for producing ______ ends.
EcoRI is a restriction enzyme known for producing ______ ends.
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Different restriction endonucleases can produce the same ______ ends.
Different restriction endonucleases can produce the same ______ ends.
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The 'N' in the sequences represents any of the four nucleotides, which are A, G, C, or ______.
The 'N' in the sequences represents any of the four nucleotides, which are A, G, C, or ______.
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The calculations about nucleotide occurrences assume a ______ arrangement of the nucleotides.
The calculations about nucleotide occurrences assume a ______ arrangement of the nucleotides.
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In practice, the assumption of equal proportions of nucleotides implies a GC content of ______%.
In practice, the assumption of equal proportions of nucleotides implies a GC content of ______%.
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Most restriction endonucleases function adequately at pH ______.
Most restriction endonucleases function adequately at pH ______.
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Restriction endonucleases usually require ______ strength for their function.
Restriction endonucleases usually require ______ strength for their function.
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All type II restriction endonucleases require ______ in order to function.
All type II restriction endonucleases require ______ in order to function.
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Adding a reducing agent like ______ stabilizes the enzyme.
Adding a reducing agent like ______ stabilizes the enzyme.
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Incorrect NaCl or Mg2+ concentrations might cause changes in the ______ of the enzyme.
Incorrect NaCl or Mg2+ concentrations might cause changes in the ______ of the enzyme.
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A suitable buffer for BglII contains ______ mM of MgCl2.
A suitable buffer for BglII contains ______ mM of MgCl2.
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Most restriction endonucleases work best at ______°C.
Most restriction endonucleases work best at ______°C.
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TaqI is a restriction enzyme from ______ aquaticus.
TaqI is a restriction enzyme from ______ aquaticus.
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The enzyme ______ is derived from Escherichia coli.
The enzyme ______ is derived from Escherichia coli.
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______ is known for producing the sticky end sequence GAATTC.
______ is known for producing the sticky end sequence GAATTC.
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BamHI is an enzyme from the organism ______.
BamHI is an enzyme from the organism ______.
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The sequence recognized by BglII is ______.
The sequence recognized by BglII is ______.
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______ produces a blunt end when cut at CAGCTG.
______ produces a blunt end when cut at CAGCTG.
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The enzyme HindIII cuts DNA at the sticky end sequence ______.
The enzyme HindIII cuts DNA at the sticky end sequence ______.
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The organism Staphylococcus aureus is associated with the enzyme ______.
The organism Staphylococcus aureus is associated with the enzyme ______.
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The sequence GCGGCCGC is recognized by the enzyme ______.
The sequence GCGGCCGC is recognized by the enzyme ______.
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Restriction endonucleases are enzymes responsible for ______ DNA.
Restriction endonucleases are enzymes responsible for ______ DNA.
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Ligases are enzymes that ______ nucleic acid molecules together.
Ligases are enzymes that ______ nucleic acid molecules together.
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Nucleases are enzymes that ______, shorten, or degrade nucleic acid molecules.
Nucleases are enzymes that ______, shorten, or degrade nucleic acid molecules.
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Polymerases are enzymes that make ______ of DNA molecules.
Polymerases are enzymes that make ______ of DNA molecules.
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Exonucleases remove nucleotides one at a time from the ______ of a DNA molecule.
Exonucleases remove nucleotides one at a time from the ______ of a DNA molecule.
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Endonucleases break internal ______ bonds within a DNA molecule.
Endonucleases break internal ______ bonds within a DNA molecule.
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DNA manipulative enzymes can be grouped into four broad classes based on the type of ______ they catalyze.
DNA manipulative enzymes can be grouped into four broad classes based on the type of ______ they catalyze.
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Some enzymes act on RNA, but this discussion will generally focus on those that act on ______.
Some enzymes act on RNA, but this discussion will generally focus on those that act on ______.
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To obtain gene B, digest with ______.
To obtain gene B, digest with ______.
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To obtain gene D, digest with ______ + Sall.
To obtain gene D, digest with ______ + Sall.
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A series of restriction digests must be performed to construct a ______.
A series of restriction digests must be performed to construct a ______.
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The sizes of the fragments produced by each restriction endonuclease must be determined by ______.
The sizes of the fragments produced by each restriction endonuclease must be determined by ______.
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Double digestions involve cutting the DNA with two restriction ______ at once.
Double digestions involve cutting the DNA with two restriction ______ at once.
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Adjusting the reaction mixture is necessary if the two digestions have different requirements for ______.
Adjusting the reaction mixture is necessary if the two digestions have different requirements for ______.
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Size markers are used to compare the sizes of the ______ produced.
Size markers are used to compare the sizes of the ______ produced.
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Information from single digestions must be supplemented by data from ______ digestions.
Information from single digestions must be supplemented by data from ______ digestions.
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Study Notes
Chapter 4: Manipulation of Purified DNA
- DNA manipulation techniques are crucial for gene cloning, DNA biochemistry, gene structure, and gene expression studies. These techniques involve cutting, joining, modifying, and copying DNA.
- Purified enzymes are essential in most DNA manipulation techniques, as they perform essential cellular functions like DNA replication, transcription, and recombination in artificial conditions.
-
Nucleases: Enzymes that cut, shorten, or degrade nucleic acids.
- Exonucleases: Remove nucleotides from the end of a DNA molecule.
- Endonucleases: Break phosphodiester bonds within a DNA molecule, either at the ends of a strand, or internally.
- Ligases: Join nucleic acid molecules together.
- Polymerases: Make copies of molecules.
- Modifying enzymes: Remove or add chemical groups. Some polymerases also possess nuclease activity (i.e., degrade DNA). There are corresponding RNA versions of these enzymes.
Restriction Endonucleases
- Crucial for precise and reproducible DNA cutting in gene cloning.
- Type II Restriction Endonucleases cut DNA at specific recognition sites.
-
Recognition sequences: Each enzyme recognizes a specific DNA sequence to cut the double-stranded DNA. Cutting locations can be blunt or sticky ends.
- Blunt ends: A single strand cut precisely at the same point on the double-helix strand.
- Sticky ends: Staggered double-helix cuts, resulting in short, complementary single-stranded overhangs. Sticky ends from different enzymes can ligate together if they are complementary.
- Recognition sequences vary in length (4 to 8 base pairs), and frequency within a DNA.
- Discovery: The discovery of restriction enzymes is a breakthrough in genetic engineering.
Ligation: Joining DNA Molecules
- Joining together DNA pieces (e.g., vector and DNA to be cloned).
-
DNA ligase: The enzyme that catalyzes this reaction. Different ligases can handle different DNA ends (e.g., blunt or sticky).
- Sticky ends ligation: More efficient: Complementary sticky ends can form temporary base-pairs aiding alignment for ligation.
- Blunt ends ligation: Less efficient, requires higher DNA concentration to increase the probability of successful ligation.
DNA Polymerases
- Enzymes that synthesize new DNA strands complementary to existing DNA or RNA templates.
- Various different DNA polymerases have specific applications in the lab.
- T4 DNA Polymerase: Fills in gaps and repairs nicks in DNA.
- Klenow Fragment: Used in DNA sequencing and filling gaps, lacking the degradation feature of DNA Polymerase I
- Taq polymerase: Thermostable polymerase commonly used in polymerase chain reaction (PCR).
DNA Modifying Enzymes
- Adding, removing chemical groups from DNA.
- Alkaline Phosphatase: Removes 5' phosphate groups.
- Polynucleotide kinase: Adds phosphate groups to 5' termini.
- Terminal deoxynucleotidyl transferase: Adds deoxyribonucleotides to 3' termini.
Restriction Digests
- Methodology for the use and characterization of restriction enzymes in the lab.
- Factors affecting restriction endonuclease activity: Temperature, pH, Mg2+ concentration.
- Methods for analyzing restriction digests: Gel electrophoresis.
Gel Electrophoresis
- Separates DNA fragments based on size.
- Important for identifying and analyzing DNA fragments cut by restriction enzymes.
- Visualization of DNA Fragments using EtBr, and other stains for visualization under UV light.
- Estimating DNA fragment sizes: Comparison with known markers, which offer an estimate of fragment lengths.
- OFAGE (Orthogonal Field Alternating Gel Electrophoresis): Improves resolution and analysis of larger DNA fragments than traditional methods.
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Description
Explore Chapter 4, focusing on the manipulation of purified DNA. This quiz covers essential DNA manipulation techniques used in gene cloning and biochemistry, including the roles of various enzymes like nucleases, ligases, and polymerases. Test your understanding of how these enzymes interact in DNA replication and modification.
