By the end of this lesson
- Describe how restriction enzymes cut DNA at specific sites.
- Explain the importance of sticky ends.
- Describe how DNA ligase joins pieces of DNA.
BIG QUESTION
How can scientists cut out one gene from millions of DNA bases?
Each restriction enzyme recognises only its own short base sequence, so it cuts in the same place every time.
Cutting with restriction enzymes
A restriction enzymeAn enzyme that cuts DNA at a specific base sequence (its recognition site), often leaving sticky ends. recognises a specific short sequence of bases, called a recognition siteA specific short sequence of bases in DNA where a restriction enzyme cuts., and cuts both strands of DNA there. Because enzymes are specific, a different restriction enzyme recognises a different sequence.
Many restriction enzymes make a staggered cut, leaving short single-stranded overhangs called sticky endsShort single-stranded sections at the ends of DNA cut by a restriction enzyme, which can pair with complementary bases on another cut piece..
Why sticky ends matter
If the same restriction enzyme is used to cut both the donor DNA (containing the wanted gene) and the vector DNA, their sticky ends have complementaryMatching in shape or base sequence, as two sticky ends with matching bases that can pair up. bases.
The complementary bases can pair up, holding the gene in position in the vector.
Joining with ligase
Base pairing alone is weak. The enzyme DNA ligaseAn enzyme that joins pieces of DNA together, such as a gene into a plasmid. joins the sugar–phosphate backbones of the two pieces, sealing the gene into the vector.
The result is recombinant DNADNA made by joining DNA from two different sources, such as a human gene in a bacterial plasmid.: DNA that contains genetic material from two different sources.
Enzymes in genetic modification
Learn what each enzyme does and what it produces.
| Enzyme | Role | Result |
|---|---|---|
| Restriction enzyme | Cuts DNA at a specific recognition site. | DNA fragments, often with sticky ends. |
| DNA ligase | Joins sugar–phosphate backbones. | A continuous recombinant DNA molecule. |
| Same restriction enzyme on both DNAs | Produces matching sticky ends. | Gene and vector ends can pair. |
Language that earns precision
- restriction enzyme
- An enzyme that cuts DNA at a specific base sequence.
- sticky ends
- Short single-stranded overhangs of unpaired bases left after a staggered cut.
- DNA ligase
- An enzyme that joins pieces of DNA together.
- recombinant DNA
- DNA made by combining DNA from two different sources.
Common misconceptions
Claim: Restriction enzymes cut DNA randomly.
Correction: They cut only at their specific recognition sequence.
Claim: Ligase cuts the gene out.
Correction: Restriction enzymes cut; ligase joins.
Claim: Any two sticky ends will join.
Correction: Ends must have complementary bases, usually by using the same restriction enzyme.
Match the answer to the command word
Always name restriction enzyme and ligase separately; do not just write “enzymes”.
Link the same restriction enzyme to complementary sticky ends and base pairing.
Recall, apply, then justify
Explain why the same restriction enzyme is used to cut the human DNA and the plasmid.
Hint
What does the enzyme leave on each piece?
Suggested marking points
- It produces the same / complementary sticky ends on both.
- So the bases of the gene and plasmid can pair up / join.
State the function of DNA ligase.
Hint
Cutting or joining?
Suggested marking points
- Joins pieces of DNA together (seals the gene into the vector).
Describe how a gene for a human protein could be removed from human DNA and joined into a bacterial plasmid.
Planning hint
Cut, cut, pair, seal.
What a strong design includes
- A restriction enzyme cuts the human DNA on either side of the gene.
- The cut leaves sticky ends.
- The plasmid is cut with the same restriction enzyme.
- This gives complementary sticky ends on the plasmid.
- The gene and plasmid are mixed so the sticky ends pair.
- DNA ligase joins the gene into the plasmid, forming recombinant DNA.