Restriction endonucleases, often called "molecular scissors," are enzymes that cut DNA at specific nucleotide sequences known as recognition sites.
Biological Role: They serve as a defense mechanism in bacteria against invading viral DNA (bacteriophages) by cutting the foreign DNA into pieces.
| Type | Characteristics | Cleavage Site |
|---|---|---|
| Type I | Complex, multi-subunit enzymes; require ATP, Mg2+, and S-adenosylmethionine. | Cuts DNA at a random site far (over 1000 bp) from the recognition sequence. |
| Type II | Simple enzymes; usually require only Mg2+. Most commonly used in lab research. | Cuts DNA exactly at or within the specific recognition sequence. |
| Type III | Intermediate complexity; require ATP and Mg2+. | Cuts DNA about 20-30 bp away from the recognition site. |
| Type IV | Target modified (usually methylated) DNA. | Cuts specifically at or near methylated recognition sites. |
Restriction mapping is a method used to map the relative locations of restriction enzyme sites on a DNA molecule.
Used for linear DNA molecules like genomic fragments. The map is created by measuring the sizes of fragments produced by single and double digestions of the DNA with different enzymes.
Used for plasmids and circular viral genomes. Because the DNA is circular, the number of fragments produced equals the number of restriction sites (e.g., 2 sites produce 2 fragments).
Vectors are DNA molecules used as vehicles to artificially carry foreign genetic material into another cell.
YACs are genetically engineered chromosomes derived from the DNA of the yeast Saccharomyces cerevisiae.
Q: What is the difference between a plasmid and a BAC?
A: A standard plasmid carries small DNA inserts (under 10 kb), while a BAC is designed to carry very large fragments (up to 300 kb) for genomic studies.
Q: Why is the Ti plasmid called a "natural genetic engineer"?
A: Because Agrobacterium naturally evolved the ability to transfer its DNA into the plant genome to manipulate the plant's machinery.