Vectors, transformation, marker genes and gene therapy - AQA A-Level Biology (Paper 2) Exam Questions

Practise 18 exam-style questions on Vectors, transformation, marker genes and gene therapy for AQA A-Level Biology (Paper 2). Write your answer as you would in the exam and get it marked in seconds against the mark scheme, point by point, with the Study Helper on hand if you get stuck.

18 questions
1–8 marks each
Instant marking
No sign-up needed

18 questions, marked as you go

Sample questions with model answers

Here are 4 of the questions in this topic, each with a model answer, an explanation of why it earns the marks and the mark scheme an examiner would use.

Question 1

2 marks

Scientists cut a plasmid with two different restriction enzymes, X and Y, which produce different sticky ends. They cut out a gene using the same two enzymes, so that the gene had an X sticky end at one end and a Y sticky end at the other end.

Suggest two advantages of using two different enzymes, rather than one enzyme, to cut the plasmid and the gene.

Model answer

1. The two ends of the cut plasmid are not complementary, so the plasmid cannot close up again without the gene, and more of the plasmids are recombinant.
2. The gene can only fit in one way round, so it is inserted in the correct orientation next to the promoter and can be transcribed.

Explanation

Sticky ends only pair with complementary sticky ends. With one enzyme, both ends of the plasmid are the same, so the plasmid can simply rejoin, and the gene can go in either way round. With two enzymes making different sticky ends, the only way for the plasmid to close is with one gene inserted the right way round.

Mark scheme

1 mark
The cut plasmid cannot rejoin / close up without the gene, because its two sticky ends are not complementary (so more plasmids contain the gene)
Max 2. List rule: if more than two answers are given, each wrong or contradictory extra answer cancels one correct answer.
1 mark
The gene can only be inserted one way round / in one orientation (e.g. the correct way round next to the promoter, so that it is transcribed correctly)
Accept 'the gene cannot be inserted backwards'.
1 mark
Two copies of the gene cannot be inserted one after the other / copies of the gene cannot join to each other the same way round
Ignore 'gene fragments cannot join to each other' (they can still join by two X ends or two Y ends).

Question 2

3 marks

Scientists inserted a gene into a plasmid that contained a gene for ampicillin resistance and a gene for tetracycline resistance. The gene was inserted into the gene for tetracycline resistance. They mixed the plasmids with bacteria that were not resistant to either antibiotic.

They spread the bacteria on plate 1, which contained agar with ampicillin. After the colonies had grown, they pressed a sterile velvet pad onto plate 1 and then onto plate 2, which contained agar with tetracycline. This transferred bacteria from each colony to the same position on plate 2. The figure shows the plates after incubation.

Give the letters of the colonies that contain the recombinant plasmid. Explain how you identified them.

Model answer

Colonies B, E and G contain the recombinant plasmid.

All the colonies on plate 1 grew on ampicillin, so they all took up a plasmid with the gene for ampicillin resistance. Colonies B, E and G did not grow on plate 2, so they are not resistant to tetracycline: the inserted gene has disrupted the gene for tetracycline resistance. Colonies A, C, D, F and H grew on both plates, so their plasmids do not contain the inserted gene.

Explanation

Replica plating transfers cells from every colony to the same position on a second plate, so colonies can be compared. The first antibiotic shows which bacteria took up any plasmid; the second shows which plasmids have lost a working resistance gene because the new gene was inserted into it. The useful colonies are the ones missing from the second plate, so scientists take them from plate 1, where they are still alive.

Mark scheme

1 mark
B, E and G
Max 3. All three, and no other letters, are needed for this mark.
1 mark
These colonies grew on ampicillin, so they contain the plasmid / were transformed, but did not grow on tetracycline
Accept 'they are resistant to ampicillin but not to tetracycline'.
1 mark
Because the inserted gene disrupted / inactivated the gene for tetracycline resistance
1 mark
Colonies A, C, D, F and H grew on both plates, so they contain plasmids without the inserted gene (both resistance genes work)

Question 3

4 marks

DNA fragments can be amplified in vitro by the polymerase chain reaction (PCR), or in vivo by inserting them into plasmids and culturing transformed bacteria.

Give two advantages of PCR compared with in vivo amplification, and two advantages of in vivo amplification compared with PCR.

Model answer

Advantages of PCR: it is much faster, producing millions of copies in a few hours; and it does not need living cells, vectors, transformation or culturing.

Advantages of in vivo amplification: long fragments, such as whole genes, can be copied; and the base sequence of the fragment does not need to be known, because no primers are needed. The transformed bacteria can also make the protein coded for by the gene.

Explanation

PCR is the in vitro method: fast, automated and able to work from tiny samples, but it needs primers (so part of the sequence must be known) and copies only fairly short fragments. In vivo cloning uses transformed host cells: slower, but it can copy long fragments without primers, and the cells can also express the gene. Give advantages of each method that compare it with the other.

Mark scheme

1 mark
PCR is faster (hours rather than days)
Max 4. Points 1 to 5 (advantages of PCR): max 2. Points 6 to 9 (advantages of in vivo): max 2. List rule: if more than two advantages of a method are given, each wrong or contradictory extra answer cancels one correct answer.
1 mark
PCR does not need living cells / vectors / transformation / culturing
Accept 'no need for restriction enzymes and ligase'.
1 mark
PCR can amplify very small (or partly degraded) samples of DNA
1 mark
PCR is automated / needs less labour
1 mark
PCR amplifies only the target sequence (between the primers)
1 mark
In vivo: fewer copying errors / mutations
Accept 'more accurate copying'. This point is credited but not required.
1 mark
In vivo: long fragments / whole genes can be copied
1 mark
In vivo: the base sequence does not need to be known (no primers needed)
1 mark
In vivo: the transformed cells can also express the gene / make the protein (or be kept as a store of the gene)

Question 4

6 marks

Describe how a gene can be inserted into a plasmid, and how bacteria that have taken up the recombinant plasmid can then be identified.

Model answer

The plasmid is cut with a restriction enzyme at a specific recognition site, and the gene is cut out with the same restriction enzyme, so both have complementary sticky ends. A promoter and terminator are added to the gene. The sticky ends pair up, and DNA ligase joins the gene into the plasmid by forming phosphodiester bonds in the sugar-phosphate backbones.

The recombinant plasmids are mixed with bacteria in ice-cold calcium chloride solution, then given a heat shock, so that some bacteria take up a plasmid (transformation). The plasmid carries a marker gene for resistance to an antibiotic, so bacteria are grown on agar containing that antibiotic and only those that took up a plasmid survive. The gene is inserted into a second marker gene (for example for resistance to a second antibiotic), so replica plating onto agar with the second antibiotic shows which colonies have the recombinant plasmid: they are the ones that do not grow.

Explanation

There are three stages: making the recombinant plasmid (same restriction enzyme, complementary sticky ends, ligase), transformation (calcium ions and heat shock), and identifying transformed cells with marker genes. Only a small proportion of bacteria take up a plasmid, and some plasmids close up without the gene, so markers are needed for both. Common errors are saying that the restriction enzyme 'cuts the gene' and that ligase forms hydrogen bonds.

Mark scheme

1 mark
The same restriction enzyme / endonuclease is used to cut the plasmid and to cut out the gene
Max 6. Accept 'restriction enzyme cuts the plasmid at a (specific) recognition site'. Reject 'restriction enzyme cuts the gene in half' or 'cuts within the gene'.
1 mark
Producing complementary sticky ends (that base pair)
1 mark
DNA ligase joins the gene and the plasmid / forms phosphodiester bonds
Reject 'ligase forms hydrogen bonds' or 'ligase makes sticky ends'.
1 mark
A promoter (and terminator) is added to the gene (so that it is transcribed)
1 mark
Bacteria are mixed with the plasmids and treated with calcium ions / heat shock / an electric field to make them take up the plasmids (transformation)
Accept 'calcium chloride and heat shock make the membrane more permeable'.
1 mark
The plasmid contains a marker gene, e.g. for antibiotic resistance / a fluorescent protein / an enzyme that produces a coloured product
Specific named marker genes are not required.
1 mark
Bacteria are grown on agar containing the antibiotic, so only bacteria that have taken up the plasmid survive / form colonies
Accept 'bacteria that glow under UV light have taken up the plasmid'.
1 mark
The gene is inserted into a second marker gene, so bacteria with the recombinant plasmid lose this marker's function (e.g. are killed by the second antibiotic in replica plating)
Accept a correct description of replica plating or of colonies that do not change colour.

Ready to try the rest? Every answer you write is marked against the mark scheme, so you can see exactly where the marks are.

Study Helper Hints first, answers after you try

Stuck on this one? Ask for a hint and I’ll point you in the right direction, without giving the answer away. Once you’ve checked your answer, I’ll show you how the marks are awarded.