Recombinant DNA Technology: Restriction Endonucleases

Study Notes

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.

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.

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

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

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

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

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)

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

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

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