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Recombinant DNA technology: isolating, inserting and expressing a gene questions
Obtaining a gene by reverse transcriptase from mRNA, by restriction endonucleases and by the gene machine; sticky ends and DNA ligase; plasmid vectors, transformation and marker genes; why transformation efficiency is so low; the polymerase chain reaction with its three temperatures and the reason for each; and gel electrophoresis.
5 original questions · 18 marks · the recombinant dna technology: isolating, inserting and expressing a gene notes · Gene regulation, genomics and biotechnology
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Explain why the same restriction endonuclease must be used to cut both the gene and the plasmid, and explain what DNA ligase then contributes.
Mark scheme
- B1 a restriction endonuclease cuts only at its own recognition sequence, and an offset cut through the two strands leaves each fragment with a short single-stranded overhang, a sticky end
- B1 using one enzyme on both means every fragment carries the same overhang, so the exposed bases of the gene are complementary to those of the cut plasmid
- B1 the overhangs pair by hydrogen bonding between complementary bases, which holds the pieces together but leaves the sugar-phosphate backbones unjoined
- B1 DNA ligase catalyses the formation of phosphodiester bonds along both strands, which makes the join permanent
A polymerase chain reaction begins with 5 molecules of double-stranded target DNA and runs for 20 cycles. Calculate the number of molecules present at the end, and calculate how long the run takes if each cycle lasts 90 seconds.
Mark scheme
- M1 the number of molecules doubles each cycle, so the number at the end is the starting number multiplied by 2 raised to the number of cycles
- M1 5 × 220 = 5 × 1 048 576
- A1 5 242 880 molecules
- A1 20 × 90 = 1800 seconds, which is 30 minutes
Well under one bacterium in a hundred takes up a plasmid, and many plasmids close on their own sticky ends without accepting an insert. Suggest why a marker gene is needed at all, and suggest how placing the gene of interest inside a gene for a fluorescent protein allows the recombinant cells to be picked out.
Mark scheme
- B1 after transformation the culture holds three populations: cells with no plasmid, cells with a self-closed plasmid and cells with the recombinant plasmid. They look identical
- B1 a marker gene gives an observable difference, so the very small proportion of cells that took up the plasmid wanted can be found without testing every colony
- B1 a plasmid that closed on itself has an intact fluorescent protein gene, so it makes the protein and its colony glows under ultraviolet light
- B1 a plasmid carrying the insert has that gene interrupted in the middle, so no functional protein is made and the colonies wanted are the ones that do not glow
State the three temperatures used in a cycle of the polymerase chain reaction, and state what each of them is for.
Explain why DNA fragments move towards the positive electrode during gel electrophoresis, and explain why the shortest fragments travel furthest.
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