Edexcel IGCSE Biology · Spec 5.12
DNA Ligase and Recombinant Plasmids
Covers distinguish restriction enzymes from DNA ligase, Explain how a recombinant plasmid is made and Describe how transformed bacteria are selected.
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DNA Ligase and Recombinant Plasmids
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Explained
Cutting DNA and joining it back together
Genetic modification needs two enzymes that do opposite jobs, and almost every mark in this topic depends on attaching the right job to the right name.
Restriction enzymes cut DNA. DNA ligase joins it. Restriction cuts, ligase ligates, and the word ligature means a tie or a binding, which is a useful hook.
Restriction enzymes
A restriction enzyme recognises a particular sequence of bases and cuts the sugar phosphate backbone at that point.
The cut is usually staggered rather than straight across, which leaves a few unpaired bases hanging off each end. Those are sticky ends, and they are sticky because their exposed bases will pair with any complementary sequence they meet.
The crucial step is to use the same restriction enzyme on both pieces of DNA. Because it cuts at the same sequence in both, the sticky ends produced are complementary to each other and will pair up.
Making a recombinant plasmid
- Cut the donor DNA containing the desired gene using a restriction enzyme.
- Cut open a bacterial plasmid using the same restriction enzyme, so the sticky ends match.
- Mix them, so the complementary sticky ends pair by base pairing.
- Add DNA ligase, which joins the sugar phosphate backbones together permanently.
The result is a recombinant plasmid, meaning one containing DNA from two sources. The plasmid is acting as a vector, the vehicle that carries the gene into the bacterium.
Transformation and selection
The plasmids are mixed with bacteria, and some take one up. A bacterium that has done so is described as transformed.
Most do not, so you need a way of finding the ones that did. Plasmids carry a marker gene alongside the useful one, often for antibiotic resistance. Grow the bacteria on a medium containing that antibiotic and only the transformed ones survive, because only they carry the resistance gene.
Those survivors are then grown in a fermenter, where they multiply and make the protein the inserted gene codes for. Human insulin is produced exactly this way.
What the mark scheme accepts and rejects
An Edexcel International GCSE Biology mark scheme awards four marks for this process: naming the restriction enzyme, saying that it cuts the donor DNA or opens the plasmid or produces sticky ends, naming ligase, and saying that it joins or inserts the DNA.
Its note is unusual and worth reading. If the function does not match the enzyme, one mark can still be awarded, for either the enzyme or the function. So naming both enzymes correctly and swapping their jobs halves the available credit rather than losing it entirely.
The examiner report on that paper is blunt about spelling. Some candidates referred to restrictive enzymes, and some to lipase rather than ligase, and the report says candidates should make sure that key technical terms are spelt accurately.
Lipase is the enzyme that digests fats. It is one letter away from ligase and belongs in an entirely different topic, and an examiner reading lipase has to mark what is written.
The same report also notes that answers referring only to the transfer of genes, without naming the specific enzymes, gained nothing at all.
Why plasmids
A plasmid is a small circular loop of DNA separate from the bacterial chromosome. Bacteria take them up naturally and copy them independently, so a gene placed in a plasmid is replicated every time the bacterium divides.
That is what makes the technique work at scale: one transformed bacterium becomes millions overnight, each carrying and expressing the inserted gene.
Spec 5.12
What you need to know
- Distinguish restriction enzymes from DNA ligase
- Explain how a recombinant plasmid is made
- Describe how transformed bacteria are selected
Active recall
Quick check
Answer each question before opening the answer.
What is the role of a restriction enzyme?
It cuts DNA at a specific recognition sequence
What is the role of DNA ligase?
It joins DNA fragments by bonding the sugar-phosphate backbone
Why is a marker gene useful?
It identifies or selects cells that received a plasmid
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