Genetic technology

• Same enzyme, complementary sticky ends, ligase makes the phosphodiester bond — and a gene is worth nothing until it is expressed.
• Same enzyme, complementary sticky ends, ligase makes phosphodiester bonds. 95, 55–65, 72 °C; 2n. DNA to the anode, short fragments furthest.

• 19.1.1 I can define — define the term recombinant DNA
• 19.1.2 I can explain — explain that genetic engineering is the deliberate manipulation of genetic material to modify specific characteristics of an organism and that this may involve transferring a gene into an organism so that the gene is expressed
• 19.1.3 I can explain — explain that genes to be transferred into an organism may be: • extracted from the DNA of a donor organism • synthesised from the mRNA of a donor organism • synthesised chemically from nucleotides
• 19.1.4 I can explain — explain the roles of restriction endonucleases, DNA ligase, plasmids, DNA polymerase and reverse transcriptase in the transfer of a gene into an organism
• 19.1.5 I can explain — explain why a promoter may have to be transferred into an organism as well as the desired gene
• 19.1.6 I can explain — explain how gene expression may be confirmed by the use of marker genes coding for fluorescent products
• 19.1.7 I can explain — explain that gene editing is a form of genetic engineering involving the insertion, deletion or replacement of DNA at specific sites in the genome
• 19.1.8 I can describe — describe and explain the steps involved in the polymerase chain reaction (PCR) to clone and amplify DNA, including the role of Taq polymerase
• 19.1.9 I can describe — describe and explain how gel electrophoresis is used to separate DNA fragments of different lengths
• 19.1.10 I can outline — outline how microarrays are used in the analysis of genomes and in detecting mRNA in studies of gene expression
• 19.1.11 I can outline — outline the benefits of using databases that provide information about nucleotide sequences of genes and genomes, and amino acid sequences of proteins and protein structures
• 19.2.1 I can explain — explain the advantages of using recombinant human proteins to treat disease, using the examples insulin, factor VIII and adenosine deaminase
• 19.2.2 I can outline — outline the advantages of genetic screening, using the examples of breast cancer (BRCA1 and BRCA2), Huntington's disease and cystic fibrosis
• 19.2.3 I can outline — outline how genetic diseases can be treated with gene therapy, using the examples severe combined immunodeficiency (SCID) and inherited eye diseases
• 19.2.4 I can discuss — discuss the social and ethical considerations of using genetic screening and gene therapy in medicine
• 19.3.1 I can explain — explain that genetic engineering may help to solve the global demand for food by improving the quality and productivity of farmed animals and crop plants, using the examples of GM salmon, herbicide resistance in soybean and insect resistance in cotton
• 19.3.2 I can discuss — discuss the ethical and social implications of using genetically modified organisms (GMOs) in food production

Precise vocabulary is part of the biology. Water moves down a water potential gradient; an active site is complementary to its substrate; enzymes are denatured, not killed; ATP releases energy when it is hydrolysed, and respiration never produces energy. Give a calculated answer to the same number of significant figures as the least precise data, or one more, with its unit. A fifth of the qualification is experimental: Papers 3 and 5 test AO3 only, and their questions may be set in contexts outside the syllabus content, so the practical work in this chapter is set out as variables, method, recording, graphs and evaluation rather than as theory.

• “The DNA runs towards the negative end, and the big pieces get furthest.” Correct DNA moves to the anode, and short fragments move furthest. DNA is negatively charged (its phosphate groups), so it moves away from the wells at the cathode (−) towards the anode (+); the gel holds long fragments back, so the shortest travel furthest.
• “DNA ligase joins the sticky ends by pairing their bases.” Correct The complementary bases pair on their own by hydrogen bonds; ligase forms phosphodiester bonds in the sugar–phosphate backbones.
• “Taq polymerase is chosen because it copies DNA quickly.” Correct Taq is chosen because it is thermostable: it is not denatured at 95 °C, so it survives every cycle and is added once.
• “A glowing cell has the gene.” Correct A glowing cell is expressing the gene: GFP fluoresces only once it has been transcribed and translated from the same promoter as the desired gene.
• “Reverse transcriptase makes mRNA from DNA.” Correct It makes DNA (cDNA) from an mRNA template; making RNA from DNA is transcription.
• “A harmful BRCA1 allele means breast cancer.” Correct It greatly raises the risk; it is not a certainty. Huntington’s disease is the example where a positive result does mean the disease.
• “Treated SCID patients pass the working allele to their children.” Correct Gene therapy in humans alters somatic cells only; eggs and sperm are unchanged, so the change is not inherited.
• “Restriction enzymes cut the DNA wherever they bind.” Repair Each restriction endonuclease cuts only at its own specific recognition sequence, because its active site is complementary to that sequence.
• “DNA ligase joins the bases of the sticky ends together.” Repair The bases pair by hydrogen bonds without help; ligase forms phosphodiester bonds in the sugar–phosphate backbone.
• “Use any restriction enzyme on the plasmid; ligase will join it.” Repair Use the same enzyme on gene and plasmid, so the sticky ends are complementary and can pair before ligase seals them.
• “Reverse transcriptase copies the gene into mRNA.” Repair It makes DNA (cDNA) from an mRNA template. RNA from DNA is transcription, by RNA polymerase.
• “The human insulin gene is cut from human DNA and put straight into bacteria.” Repair A human gene contains introns, which bacteria cannot remove; cDNA made from spliced mRNA, or a synthesised gene, is used.
• “The promoter makes the gene work faster.” Repair The promoter is where RNA polymerase binds; without one the host recognises, the gene is not transcribed at all.
• “GFP shows the cell contains the gene.” Repair GFP fluoresces only when it has been made, from the same promoter as the desired gene, so a glowing cell is expressing the gene.
• “In PCR the primers bind at 95 °C and the strands separate at 72 °C.” Repair Separate at about 95 °C (hydrogen bonds break); primers anneal at about 55–65 °C; Taq extends at about 72 °C.
• “Taq polymerase is used because it works at a higher speed.” Repair It is thermostable: it is not denatured at 95 °C, so it survives every cycle and is added once.
• “After 10 cycles of PCR there are 20 copies.” Repair Copies double each cycle: 210 = 1024 from one molecule, not 2 × 10.
• “DNA is attracted to the cathode because it is negative.” Repair Negative DNA moves away from the cathode (−), where the wells are, towards the anode (+).
• “Large fragments move further because they are heavier.” Repair Short fragments move further; the gel’s mesh holds long ones back. Charge per unit length is the same for all.
• “Gene therapy for SCID cures the patient’s future children too.” Repair Somatic gene therapy changes body cells only; eggs and sperm are unchanged, so the change is not inherited.
• “Pests become immune to Bt cotton.” Repair Resistance is not immunity: pests with a resistance allele survive and breed, so the allele is selected and becomes more common (17.2.4).

• Read the command word before you decide how much to write. This syllabus has seventeen of them: assess, calculate, comment, compare, contrast, define, describe, determine, discuss, explain, give, identify, outline, predict, sketch, state and suggest. State, give and identify want a fact and nothing more. Define wants a precise meaning. Outline: set out the main points. Describe: state the points of a topic, or give its characteristics and main features. Explain: set out purposes or reasons, make the relationships between things clear, say why and/or how and support with relevant evidence — a describe-level answer to an explain question is incomplete. Compare: identify or comment on similarities and/or differences; contrast: differences only. Discuss wants the issue written about in depth, in a structured way; assess wants an informed judgement. Suggest asks you to apply what you know to a situation where there is a range of valid responses, making proposals or putting forward considerations.
• Interleave with the chapters that use this one. Topic 19 is the last topic, so interleave backwards: with chapter 6, re-answer “why does cDNA have no introns?”; with chapter 16, “what is the chance a child of a heterozygous Huntington’s parent inherits the allele?”; with chapter 17, “why do refuges slow resistance in Bt cotton?”; with chapter 18, “how can GM crops affect farmland biodiversity?”. Recalling a topic inside a new context is worth more than another pass over this chapter on its own; at A Level, Paper 4 assumes the whole of the AS content, so nothing here is ever finished with.

• Cambridge International AS & A Level Biology 9700 has five components. Topic 19 is A Level content, so it is examined in Papers 4 and 5. A Level content: examined in Paper 4 (A Level structured, which also requires the AS content) and, as practical context, Paper 5. AS Level candidates take Papers 1, 2 and 3; A Level candidates take all five, either staged over two years (Papers 1–3 in year one, Papers 4 and 5 in year two) or together in one series. Examinations are available in the June and November series, and in March in India.
• Across both the AS Level and the A Level the assessment objectives are weighted AO1 40% (knowledge and understanding), AO2 40% (handling, applying and evaluating information) and AO3 20% (experimental skills and investigations). AS candidates are graded a–e; A Level candidates A*–E. There is no data booklet in Biology. At A Level, the statistical formulae (Hardy–Weinberg, the Lincoln index, Simpson’s index, standard deviation, standard error, 95% confidence intervals, the χ² test, the t-test, and Pearson’s and Spearman’s correlation) are printed in a question when it needs them, and so are the tables of critical values; degrees of freedom you must work out yourself. Everything else — magnification, surface area to volume, RQ, Rf, rates — you must recall, and this chapter says which is which.
• Topic 19 is not in Paper 1. Paper 4 structured questions ask you to define recombinant DNA, explain the role of each enzyme and why a promoter or marker is needed, describe PCR or gel electrophoresis step by step, outline microarrays, screening and gene therapy, and discuss the ethics — often as an extended answer such as the production of insulin.
• A drawing of a gel with a ladder to read (distance against lg of length); a PCR temperature trace; copies after n cycles (start × 2n, recalled); a two-colour microarray to interpret; a pedigree or screening result, with probabilities as fractions, decimals or percentages; yield data from a GM trial.
• The topic names no Paper 3 practical; gel electrophoresis is the closest investigation. Independent variable: fragment length from a ladder (or voltage); dependent variable: distance to the band centre, to 0.5 mm; standardised: gel, buffer, voltage, time and volume loaded; control: a dye-only well; main error: judging the centre of a blurred band. In Paper 5, a t-test on GM and non-GM yields.
• Read the command word before you decide how much to write. This syllabus has seventeen of them: assess, calculate, comment, compare, contrast, define, describe, determine, discuss, explain, give, identify, outline, predict, sketch, state and suggest. State, give and identify want a fact and nothing more. Define wants a precise meaning. Outline: set out the main points. Describe: state the points of a topic, or give its characteristics and main features. Explain: set out purposes or reasons, make the relationships between things clear, say why and/or how and support with relevant evidence — a describe-level answer to an explain question is incomplete. Compare: identify or comment on similarities and/or differences; contrast: differences only. Discuss wants the issue written about in depth, in a structured way; assess wants an informed judgement. Suggest asks you to apply what you know to a situation where there is a range of valid responses, making proposals or putting forward considerations.