Restriction enzymes are molecular scissors that bacteria naturally use to cut invading viral DNA at specific sequences. In biotechnology, scientists use these enzymes to cut DNA in predictable places and isolate useful genes. This makes it possible to build recombinant DNA, which combines DNA from different sources.
Recombinant DNA is important in medicine, agriculture, research, and the production of proteins such as insulin.
A common method uses a circular bacterial plasmid as a DNA carrier, also called a vector. The plasmid and the foreign DNA are cut with the same restriction enzyme so their ends can match. DNA ligase then seals the sugar phosphate backbone, creating a stable recombinant plasmid.
When the plasmid enters a bacterial cell, the inserted gene can be copied and sometimes expressed as a protein.
Key Facts
- Restriction enzymes cut DNA at specific recognition sites, often 4 to 8 base pairs long.
- Many restriction enzymes make sticky ends, which are single-stranded overhangs that can base-pair with matching DNA ends.
- Complementary base pairing follows A pairs with T and C pairs with G.
- DNA ligase joins DNA fragments by forming phosphodiester bonds in the sugar phosphate backbone.
- Recombinant DNA contains genetic material from two or more different sources.
- A plasmid vector often includes an origin of replication, a selectable marker, and one or more restriction sites.
Vocabulary
- Restriction enzyme
- A protein that cuts DNA at a specific nucleotide sequence called a recognition site.
- Recognition site
- A short DNA sequence that a restriction enzyme identifies and cuts.
- Sticky end
- A single-stranded DNA overhang produced by some restriction enzyme cuts that can pair with a complementary overhang.
- DNA ligase
- An enzyme that seals breaks in DNA by joining adjacent nucleotides with phosphodiester bonds.
- Plasmid vector
- A small circular DNA molecule used to carry a gene into a cell for copying or expression.
Common Mistakes to Avoid
- Using different restriction enzymes without checking end compatibility is wrong because the plasmid and insert may not have matching sticky ends.
- Forgetting DNA ligase is wrong because base pairing alone does not permanently seal the DNA backbone.
- Assuming every restriction enzyme makes sticky ends is wrong because some enzymes make blunt ends with no overhangs.
- Ignoring the orientation of the inserted gene is wrong because a gene placed backward may not be expressed correctly from the plasmid promoter.
Practice Questions
- 1 A restriction enzyme recognizes a 6 base pair sequence. If bases are randomly distributed, about how often would this site appear in a DNA molecule, in base pairs?
- 2 A plasmid is 4000 base pairs long and a gene insert is 1200 base pairs long. What is the total length of the recombinant plasmid after the insert is ligated into the plasmid?
- 3 A plasmid and a gene insert are cut with the same restriction enzyme, producing matching sticky ends. Explain why this helps the insert join the plasmid and why ligase is still needed.