Genetic Engineering & CRISPR

Q1. (3 marks) — 1 mark each

• (a) Recombinant DNA: DNA formed by joining together DNA fragments from two different sources/organisms. (1)
• (b) Restriction enzyme: an enzyme (endonuclease) that cuts DNA at a specific recognition sequence. (1)
• (c) Plasmid vector: a small circular DNA molecule (from bacteria) used to carry/introduce foreign DNA into a host cell. (1)

Q2. (5 marks)

• (a) A sequence that reads the same $5'\to3'$ on both complementary strands. (1)
• (b) Sticky ends have short single-stranded overhangs (1) that are complementary and base-pair with any DNA cut by the same enzyme, allowing easy joining/higher ligation efficiency. (1)
• (c) Cut between G and A on each strand: $5'\text{-G} \downarrow \text{AATTC-}3'$ $3'\text{-CTTAA} \uparrow \text{G-}5'$ Correct base-pairing/complementary strand (1); correct staggered cut position giving AATT overhangs (1).

Q3. (3 marks) — 1 mark each

• (a) Ligase joins/seals the sugar–phosphate backbone (forms phosphodiester bonds) between insert and vector. (1)
• (b) Acts as a selectable marker — allows only transformed bacteria to survive on antibiotic medium. (1)
• (c) Host cell replicates/copies the plasmid and expresses the gene (provides cellular machinery). (1)

Q4. (4 marks)

• Bacteria are made competent (e.g. treated with $\text{CaCl}_2$ / heat shock or electroporation) (1)
• so they take up the plasmid DNA from solution. (1)
• Grown on selective medium with antibiotic — only cells with plasmid survive. (1)
• Blue/white screening (or marker gene disruption) distinguishes recombinant from non-recombinant plasmids. (1)

Q5. (7 marks)

• (a) Denaturation ~94–95 °C; Annealing ~50–65 °C; Extension ~72 °C. (3, one each)
• (b) Copies $= 2^{n} = 2^{10} = 1024$. (formula 1, answer 1)
• (c) Taq is a DNA polymerase that synthesises new strands (1); it is thermostable, so survives the high denaturation temperature without denaturing itself. (1)

Q6. (4 marks)

• qPCR (quantitative/real-time PCR): measures the amount of DNA in real time as it is amplified (1); application e.g. measuring viral load / quantifying gene copy number. (1)
• RT-PCR (reverse transcriptase PCR): converts RNA into cDNA using reverse transcriptase before amplification (1); application e.g. detecting RNA viruses / studying gene expression. (1)

Q7. (4 marks)

• (a) DNA is negatively charged due to its phosphate groups (1), so it migrates towards the positive electrode (anode). (1)
• (b) Smaller fragments migrate further/faster than larger fragments. (1)
• (c) e.g. forensic identification / paternity testing. (1)

Q8. (5 marks)

• (a) The gRNA has a sequence complementary to the target DNA (1) and directs Cas9 to the correct location by base-pairing. (1)
• (b) Cas9 is an endonuclease that cuts (creates a double-strand break in) the DNA. (1)
• (c) PAM = Protospacer Adjacent Motif, a short sequence (e.g. NGG) next to the target (1); Cas9 requires it to bind and cut / provides specificity. (1)

Q9. (2 marks)

• Knockout: a gene is disabled/inactivated (loss of function). (1)
• Knock-in: a new/functional gene or sequence is inserted at a specific site. (1)

Q10. (4 marks)

• (a) Base editing chemically converts one base to another (e.g. C→T) directly (1) without making a double-strand break / uses a modified "dead" Cas9. (1)
• (b) Any two: heritable changes passed to future generations; consent of unborn/future individuals; safety/off-target effects; equity/access ("designer babies") — (1 each, max 2).

[
  {"claim":"PCR from 1 copy after 10 cycles gives 1024 copies","code":"n=10; result = (2**n == 1024)"},
  {"claim":"PCR doubling formula: 5 cycles from 1 copy = 32","code":"result = (2**5 == 32)"},
  {"claim":"Taq extension temperature 72 within denaturation-below range","code":"result = (72 < 95 and 72 > 65)"}
]