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Restriction enzymes cut RNA at specific sequences.
False
The first genetically modified organisms were developed in 1974.
False
Type II restriction enzymes cut DNA randomly upstream or downstream of the recognition site.
False
Transgenic organisms are formed by adding foreign RNA to an organism's genome.
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EcoRI and BamHI are examples of sticky-end generating restriction enzymes.
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Type I restriction enzymes cut DNA within the recognition site.
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In 1994, genetically modified food began to be sold commercially.
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Type V restriction enzymes require a specific guide RNA to cut DNA.
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Type IIP subgroups cut away from the recognition site.
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Neoschizomers recognize the same sequence but cut it differently.
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Isoschizomers cut DNA at different locations instead of the same location.
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Type IIE requires two recognition sites, and one acts as an allosteric effector.
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Type IIF cuts at both sites as a dimer after bringing the two regions together by looping the DNA.
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Restriction mapping is used to show the positions of recognition sequences for different enzymes.
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The ligation of compatible sticky ends produces a new sequence that can be digested by any of the different restriction enzymes.
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Enzyme A produces fragments of two sizes when digesting a specified DNA fragment.
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EcoRI and BamHI both produce sticky ends when they cut DNA fragments.
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Blunt end ligation is an efficient process for joining DNA fragments.
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T4 DNA ligase requires NAD+ as an energy source for its ligation activity.
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The efficiency of ligation with E. coli ligase is high compared to T4 DNA ligase.
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Ligases can only catalyze the formation of phosphodiester bonds between sticky ends.
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DNA topoisomerases possess both nuclease and ligase activities, enabling efficient blunt end ligation.
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The digestion of DNA fragments with BamHI and EcoRI produces the same number of total fragments.
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Isoschizomer restriction enzymes produce the same sticky ends when cutting DNA.
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Topoisomerase cuts DNA at the sequence CCCTT, which is present multiple times in the plasmid.
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After cutting the plasmid, topoisomerase enzymes remain covalently bound to the resulting sticky ends.
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The insert produced by cutting with a restriction enzyme is treated with alkaline phosphatase to add terminal 5' phosphates.
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Cloning with two sticky ends is characterized by high vector self-ligation.
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Directional cloning is lost when using blunt ends for cloning.
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Addition of linkers involves adding single-stranded oligonucleotides to blunt ends without the need for digestion.
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Homopolymer tailing involves adding homopolymers to the 5' ends of DNA molecules.
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Cloning with one sticky end and one blunt end maintains directional cloning.
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Study Notes
Molecular Biology
- The study of the molecular nature of the gene and its mechanisms of gene replication, transcription, and translation.
Molecular Genetics
- Relies on genetic engineering to modify organisms by adding foreign DNA, creating transgenic organisms.
Chronology of Genetic Engineering
- 1973: First genetically modified (GM) bacteria created.
- 1974: Genetically modified mice created.
- 1982: First commercial development of genetically modified organisms (GMOs) with insulin-producing bacteria.
- 1994: Genetically modified food was introduced for sale.
Restriction Enzymes (Restriction Endonucleases)
- Enzymes produced naturally by bacteria, cutting DNA at specific base sequences known as recognition sites.
- Recognition sites are typically 4-8 base pairs long and palindromic.
Types of Restriction Enzymes
- Type I: Cut DNA randomly upstream or downstream of the recognition site.
- Type II: Cut DNA within or near the recognition sequence.
- Type III: Cut DNA 20-30 bp downstream of the recognition site.
- Type IV: Recognize modified (methylated) DNA.
- Type V: Artificial restriction enzymes generated by fusing a DNA binding domain to a nuclease domain.
Type II Subgroups
- Type IIP: Cut at the recognition site.
- Type IIS: Cut away from the site.
- Type IIB: Requires two recognition sites and cuts on the outside.
- Type IIE: Requires two recognition sites, one acting as an allosteric effector.
- Type IIF: Requires two sites and cuts at both sites as a tetramer, looping DNA.
- Type II: Acts as a monomer instead of a dimer.
Concepts Related to Restriction Enzymes
- Neoschizomers: Different restriction enzymes recognize the same sequence but cut it differently.
- Isoschizomers: Different restriction enzymes recognize the same sequence and cleave at the same location.
- Isocaudomer: Different restriction enzymes recognize and cleave different sequences, generating compatible or complementary sticky ends.
Restriction Mapping
- A technique to construct a map of the relative positions of recognition sequences for different restriction enzymes in a DNA molecule.
Ligases
- Enzymes that catalyze the formation of phosphodiester bonds, joining DNA segments together.
Types of Ligases
-
Bacterial DNA ligase:
- Requires NAD+ or NADP+ as an energy source.
- Low ligation efficiency, ineffective in joining blunt ends.
-
Bacteriophage Ligase:
- T4 DNA ligase requires ATP as an energy source.
- High ligation efficiency, capable of joining both sticky and blunt ends.
Blunt End Ligation with DNA Topoisomerase
- Efficient method for blunt end ligation.
- DNA topoisomerases have both nuclease and ligase activities, cutting DNA at specific sequences and remaining bound to the blunt ends.
Cloning with Different End Types
-
Sticky Ends:
- Requires compatible sticky ends.
- Directional cloning.
- Efficient ligation of insert and vector.
- Low vector self-ligation.
- Recognition sites of ligated restriction enzymes remain intact.
-
Different But Compatible Sticky Ends:
- Requires compatible sticky ends.
- Directional cloning.
- Efficient ligation of insert and vector.
- Low vector self-ligation.
- Original recognition sites may be destroyed after ligation.
-
Sticky and Blunt Ends:
- Directional cloning maintained.
- Ligation of blunt ends might be less efficient.
-
Blunt Ends:
- Ends are compatible.
- End sequences are modified.
- Directional cloning lost.
- Ligation might be less efficient.
- High vector self-ligation.
Conversion of Blunt Ends to Sticky Ends
-
Linkers:
- Double-stranded oligomers containing a recognition sequence for a specific restriction enzyme are added to blunt ends and then digested.
-
Adaptors:
- Short, double-stranded sticky-ended oligonucleotides added to blunt ends, requiring no further digestion.
-
Homopolymer Tailing:
- Enzyme terminal transferase (TdT) adds homopolymers of dA, dT, dG, or dC to the 3' ends of DNA molecules.
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Description
Test your knowledge on molecular biology and the timeline of genetic engineering breakthroughs. Explore topics like restriction enzymes and their types, as well as the principles of molecular genetics. This quiz covers essential concepts and historical milestones in the field.
