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.
Understanding Biology: Restriction Enzymes and Recombinant DNA
Restriction enzymes recognise short DNA patterns because their protein shapes fit particular base sequences. Many recognition sites read the same in opposite directions when each DNA strand is read in its own direction. This symmetry helps the enzyme bind correctly to double stranded DNA.
Different enzymes cut at different positions within or near their recognition sites. Some leave short exposed ends, while others cut straight across both strands.
Exposed ends make it easier to join selected pieces, but blunt ends can be joined too. Blunt end joining is usually less efficient because there is no temporary base pairing to hold the fragments together.
A DNA sample contains millions of base pairs, so choosing the right enzyme matters. Scientists first examine the sequence of the plasmid and the DNA fragment they want to insert. The chosen enzyme should cut the plasmid only once, otherwise the carrier may break into several pieces.
It should cut on either side of the target gene without cutting through the gene itself. Sometimes two different enzymes are used, one at each end of the fragment. This gives two unlike ends.
The insert can then enter the plasmid in only one direction. Direction matters when a gene must be read by the cell in the correct orientation.
Joining DNA is only one stage of making a useful bacterial strain. Not every bacterial cell takes up a plasmid, and not every plasmid contains the desired insert. A selectable marker helps identify cells that received a plasmid.
For example, cells may survive on a growth plate containing an antibiotic only if they carry a resistance gene on the plasmid. A second test is needed to tell whether the plasmid contains the insert.
Scientists may use DNA sequencing, a test that reads the base order, or they may cut the recovered plasmid with enzymes and check the fragment sizes. These checks are essential because DNA can rejoin without the target fragment or can contain a fragment in the wrong direction.
For a transferred gene to make a protein, more than the coding sequence may be needed. Bacterial cells must have instructions that tell them where to begin copying the gene into RNA. These control sequences are called promoters.
A gene from a human cell may need a bacterial promoter before bacteria can use it well. Bacteria also cannot remove introns, which are noncoding sections found in many human genes. For this reason, scientists often start with a DNA copy made from mature human RNA.
This copy contains the protein instructions without introns. Students should keep separate the ideas of cutting DNA, copying DNA, and expressing a gene. They are linked steps, but each depends on different cell machinery and different evidence.
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.