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Questions and Answers
Restriction enzymes are used to ______ a DNA segment.
Restriction enzymes are used to ______ a DNA segment.
digest
Gel ______ is used to separate pieces of DNA according to their size.
Gel ______ is used to separate pieces of DNA according to their size.
electrophoresis
A ______ is a population of identical organisms derived from a single parental organism.
A ______ is a population of identical organisms derived from a single parental organism.
clone
Plasmids are pieces of ______ DNA that are extra chromosomal.
Plasmids are pieces of ______ DNA that are extra chromosomal.
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A ______ vector is a vector that can propagate in two different host species.
A ______ vector is a vector that can propagate in two different host species.
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Prokaryotes, such as bacteria, use ______ enzymes to destroy foreign genetic material in defence.
Prokaryotes, such as bacteria, use ______ enzymes to destroy foreign genetic material in defence.
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Restriction endonucleases target ______ DNA as substrate with high precision to specific nucleotide sequences.
Restriction endonucleases target ______ DNA as substrate with high precision to specific nucleotide sequences.
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Type I ______ endonucleases require ATP to hydrolyse DNA and cause chemical modifications of DNA by methylation.
Type I ______ endonucleases require ATP to hydrolyse DNA and cause chemical modifications of DNA by methylation.
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Eco RI is a type of ______ endonuclease that recognizes the hexanucleotide sequence GAATTC.
Eco RI is a type of ______ endonuclease that recognizes the hexanucleotide sequence GAATTC.
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The method of mapping an unknown segment of DNA by breaking it into pieces and then identifying the locations of the breakpoints is called ______.
The method of mapping an unknown segment of DNA by breaking it into pieces and then identifying the locations of the breakpoints is called ______.
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Study Notes
Restriction Enzymes
- Restriction enzymes, also known as restriction endonucleases, are enzymes primarily isolated from bacteria that cleave double-stranded DNA (dsDNA).
- They are used by prokaryotes to destroy foreign genetic material, such as viruses, in defense.
Characteristics of Restriction Endonucleases
- Restriction endonucleases target dsDNA with high precision to specific nucleotide sequences (target sequence).
- Some restriction endonucleases are isoschizomers, which recognize and cleave the same target sequence of dsDNA.
- These enzymes generate fragments with 5’-PO4 and 3’-OH endpoints.
Classification of Restriction Endonucleases
- Type I restriction endonucleases:
- Require ATP to hydrolyze DNA
- Cause chemical modifications of DNA by methylation
- Cleave DNA randomly
- Type II restriction endonucleases:
- Do not require ATP for hydrolytic activity
- Do not modify DNA by methylation
- Recognize and cleave dsDNA at specific target sequences
- Recognition sequence consists of 4-6 base-pairs, with a twofold axis of symmetry
- Can be cleaved into blunt ends or sticky-ends
- Example: EcoRI, which recognizes the hexanucleotide sequence GAATTC
- Type III restriction endonucleases:
- Require ATP to hydrolyze DNA
- Cause chemical modification of DNA by methylation
- Recognize and cleave dsDNA at specific target sequences
Naming of Restriction Endonucleases
- Restriction endonucleases are written in italics (3 letter code)
- The first letter (Capital letter) indicates the genus of the organism
- The second and third letter represents an abbreviation of the species
- Prokaryotes with more than one restriction enzyme are also provided a number
Restriction Mapping
- A method used to map an unknown segment of DNA by breaking it into pieces and then identifying the locations of the breakpoints
- Relies on the use of restriction enzymes
- After a DNA segment has been digested, the resulting fragments can be examined using gel electrophoresis, which separates pieces of DNA according to their size
Cloning and Expression Vectors
- Why genetically modify organisms?
- To create disease, drought, and pest resistant crops that are nutritionally enhanced
- To create modified animal models for research regarding cancer, obesity, heart diseases, etc.
- To create modified mosquitoes to prevent diseases such as malaria
- To produce drugs, such as insulin, hormones, vaccines, and anti-cancer drugs
Cloning Vectors
- What does it mean to clone?
- A clone is a population of identical organisms derived from a single parental organism
- Plasmids and bacteriophages are used as cloning vectors
- Vector is used to indicate a vehicle or carrier of a gene/genes
Plasmids
- Extra chromosomal DNA – circular
- Bacteria often have plasmids, which are circular loops of DNA
- Bacteria can also take in new plasmids
- Plasmids have three common features:
- Replicator (origin of replication)
- Selectable marker (gene referring resistance to an antibiotic)
- Cloning site (sequence of one or more restriction sites)
Recombinant/Chimeric Plasmids
- Foreign DNA fragment inserted into plasmid using RE digestion or other cloning method
- The polylinker or MCS (multiple cloning sites) contains various restriction sites and long stretches of nucleotides enabling transcription and translation
Bacteriophages
- Bacteria infecting viruses
- Plasmids can only accommodate a fragment of up to 10 kb
- Bacteriophages, such as bacteriophage λ, can be used to clone fragments of up to 40 kb
- These recombinant bacteriophages replicate in E. coli as plasmids
Shuttle Vectors
- A shuttle vector is a vector (usually a plasmid) constructed so that it can propagate in two different host species
- Therefore, DNA inserted into a shuttle vector can be replicated in two different host species
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Description
This quiz covers the characteristics and functions of restriction endonucleases, enzymes isolated from bacteria that cleave dsDNA in defense against foreign genetic material.
