3.8.4 is the laboratory toolkit. Specific tools cut DNA at named recognition phrases. Other tools join the pieces back together. Specialised machines copy a sequence inside a bacterium or outside any cell, find specific sequences by complementary matching, and sort the fragments by size and charge.
Read this topic as an analogy.
Imagine a workshop equipped to manipulate long printed documents: cut, paste, copy, stamp, sort, and replace specific passages. Each tool in the workshop does one thing precisely. A workshop manager combines the tools to produce particular outputs: a stitched-together hybrid document, a stack of copies of one section, a tagged search through a master text for a particular phrase, a sorted set of slips of paper from longest to shortest.
The scissors are calibrated to cut only where a specific recognition phrase appears in the text. The phrase reads the same forwards and backwards (palindromic), so the cut is precise and reproducible. The scissors cut on a stagger, leaving short ragged edges on either side. Edges from the same scissors interlock with each other because the staggered cuts produce complementary patterns. The adhesive operator brushes the ragged edges of two documents together, the edges hold by their matching pattern, and a permanent bond seals the join. A small circular bound document (a plasmid) is now stitched together with a new paragraph inside it, and the bound document goes into a working copy office (a bacterium) that has been persuaded to leave its doors briefly open.
The photocopier doubles its input every cycle. Feed it once and walk away with two copies; feed those back and walk away with four; after n cycles you have 2^n. It works in three stages each cycle: heat enough to peel the two sheets of a document apart, cool enough for short paragraph-locators to attach, then warm to the photocopier's optimum for the copying mechanism itself. The mechanism is designed to keep working at the temperature where the peeling happens, so the whole cycle can run uninterrupted.
The stamp operator carries a stamp shaped like a specific phrase. The stamp adheres only where the same phrase appears in the document; everywhere else it slides off. A coloured ink on the stamp shows where the phrase was. The sorting tray uses a current that pulls slips of paper through a sieve. Small slips slip through fast and travel far; large sheets snag in the mesh and stay near the start. Two features matter for how the slips end up: the size of the paper, and the current that pulled it.
When the source is a working copy (a printed transcript with marginalia removed already, ready for the press), there is a tool that reads it back to a master copy. The master copy can then be filed alongside the working copies and used wherever the original was needed. That same workshop, finally, can paste a corrected paragraph into a person's own master document where the original paragraph was faulty. The risk is the courier: the small parcel carrying the new paragraph may itself be recognised by the office mail room and rejected, regardless of the value of the paragraph inside it.
Mapping back to formal vocabulary. Scissors are restriction endonucleases cutting at recognition sites with palindromic sequences, leaving sticky ends. The adhesive is DNA ligase forming phosphodiester bonds; the bound document is the plasmid. The photocopier is PCR doubling through 2^n cycles with Taq polymerase. The stamp is the DNA probe, single-stranded with complementary bases that hybridise to the target. The sorting tray is gel electrophoresis, separating fragments by mass and by the negative charge of the phosphate backbone. The paragraph reading the working copy back to the master is reverse transcriptase producing cDNA. The corrective paragraph paste is gene therapy. Use the formal terms in the exam; the workshop is for understanding.
A gene of interest can be obtained by reverse transcriptase, by restriction endonuclease, or by a gene machine.
Before a gene can be cloned, copied by PCR, or used in any of the downstream applications, a copy of the gene has to be obtained. AQA names three routes. Each starts from a different source, uses a different tool, and produces a slightly different product.
Reverse transcriptase, found in certain viruses, synthesises single-stranded complementary DNA (cDNA) from an mRNA template. A second strand follows to give double-stranded cDNA. The advantage for bacterial expression: cDNA contains only the exons, because the mRNA was already spliced — bacteria cannot splice introns themselves.
Reverse transcriptase converts mRNA into cDNA. Don't reverse the direction (DNA into mRNA) and don't write tRNA as the product. Where RT-PCR is used, the existing DNA in the sample must be
hydrolysedfirst, so it is not amplified alongside the cDNA. AQA rejectssingle-stranded by heating,nucleotide source, andDNA helicase denaturedas substitutes for the hydrolyse-and-remove explanation.
Bacterial enzymes that cut double-stranded DNA at short palindromic recognition sites, typically six base pairs long. The cuts are staggered, leaving short single-stranded overhangs called sticky ends on either side of the cut. Sticky ends from the same enzyme are complementary and can base-pair across the cut.
Where the amino acid sequence of the desired protein is known, the genetic code is used in reverse to design a corresponding DNA sequence. The gene machine then synthesises that DNA chemically. The route does not require access to a cell expressing the gene.
Write
restriction endonuclease. AQA rejectsenzymealone and rejectsendonucleasealone;restriction enzymeis accepted. Writerecognition site,palindromic sequence, orrestriction sitefor the cut location; AQA rejectsspecific sitealone. Heat denatures DNA by separating the strands; it does not cut the phosphodiester backbone. Only restriction endonucleases cut.
The gene is inserted into a plasmid, the plasmid into a bacterium, and gene markers identify successful uptake.
Once a copy of the gene is in hand, it is inserted into a vector, usually a bacterial plasmid. Plasmids are small, circular, double-stranded DNA molecules that exist separately from the bacterial chromosome and replicate independently. A plasmid carrying an inserted gene from a different organism is a recombinant DNA molecule.
- The same restriction enzyme cuts both the plasmid and the source DNA. Sticky ends on the plasmid and the insert are complementary because both came from the same recognition site.
- Sticky ends anneal by complementary base pairing. Hydrogen bonds form between the matching bases across the cut.
- DNA ligase joins the sugar-phosphate backbones by phosphodiester bonds. The plasmid is sealed back into a closed circle containing the insert.
- A complete gene construct includes a promoter at the start and a terminator at the end. The promoter allows RNA polymerase to begin transcription; the terminator is where transcription stops.
Bacterial cells do not readily take up exogenous DNA. Electroporation uses brief electrical pulses to create transient pores in the membrane. Calcium chloride treatment, often combined with a rapid temperature shift from 0 °C to about 40 °C, also increases membrane permeability. The recombinant plasmid enters through the temporarily permeable membrane.
Antibiotic resistance markers let transformed bacteria survive on antibiotic-containing media; insertional inactivation of a second antibiotic resistance gene distinguishes recombinant carriers from re-circularised plasmids. Fluorescent markers emit light under specific wavelengths. Enzyme markers produce a colour change on a specific substrate.
Restriction endonuclease cuts the plasmid. DNA ligase joins the sticky ends. AQA rejects
ligase creates sticky endsandrestriction enzyme cuts the gene— the gene has already been isolated; the plasmid is what is being opened. Ligase formsphosphodiesterbonds, not hydrogen bonds; hydrogen bonds are between complementary base pairs in the annealing step. Writepromoterandterminatorfor the regulatory regions; AQA rejectsstart codonfor promoter andstop codonfor terminator.
PCR amplifies a DNA sequence exponentially through three temperature-controlled steps.
PCR amplifies a specific DNA sequence outside any living cell, through repeated cycles of denaturation, primer annealing, and enzymatic extension. The reaction mixture contains the DNA template, two short DNA primers flanking the target sequence, free nucleotides, and Taq polymerase.
- Denaturation at about 95 °C. Hydrogen bonds between the two DNA strands break; the double helix separates into two single-stranded templates.
- Annealing at 50–65 °C. The primers attach to their complementary sequences on each template strand by base pairing.
- Extension at about 72 °C. Taq polymerase synthesises a new complementary strand from the free nucleotides, joining them by phosphodiester bonds.
Heat breaks the hydrogen bonds between complementary base pairs in denaturation. Taq polymerase forms
phosphodiesterbonds along the new strand in extension. AQA rejectshydrogen bondsas the bond polymerase forms.
PCR amplification is exponential. Write 2^n in the working before any arithmetic — that secures the method mark even if the final number is wrong.
Linear multiplication is the consistently dropped mark. After 30 cycles you do not get "30 copies"; you get roughly 2³⁰, about a billion copies, from a single starting molecule. Show 2^n in the working.
Taq polymerase is isolated from the thermophilic bacterium Thermus aquaticus. It is used because it remains functional at the temperature where the strands are denatured. PCR does not stop because of a stop codon — stop codons operate in translation, not DNA replication.
AQA rejects
stop codonas the reason PCR stops. PCR stops because the primers or the free nucleotides are exhausted, or because Taq polymerase eventually denatures after enough heat cycles.
DNA probes detect specific sequences by complementary hybridisation.
A DNA probe is the tool for locating a specific target sequence within extracted DNA. The probe is short, single-stranded, and base-sequence-complementary to the target. Both structural features are required in the credited definition.
The structural definition requires both components. Label type, marker gene, and detection method are not structural features and are not credited as substitutes.
- The sample DNA is denatured to produce single strands.
- The labelled probe is added; it hybridises to its complementary target sequence by complementary base pairing.
- The label marks the position of the target. Radioactive labels are detected by autoradiography; fluorescent labels are detected under UV or specific wavelengths.
labelled probealone is not a definition. AQA rejects it for the structural mark; the credited definition requires single-stranded plus complementary bases. A DNA probe is not a marker gene either — marker genes confirm that transformation has occurred (§3.2); probes locate specific target sequences in extracted DNA. The two tools are distinct and not interchangeable in mark schemes.
Genetic fingerprinting reads VNTR patterns separated by mass and charge on a gel.
Genetic fingerprinting exploits non-coding regions called variable number tandem repeats (VNTRs). At specific chromosomal loci, short DNA sequences are repeated a variable number of times in different individuals. The combination of repeat counts across multiple loci is statistically unique to each person.
- Extract DNA from the sample.
- Amplify the VNTR loci by PCR to produce enough material to visualise.
- Load the amplified fragments into wells at one end of an agarose gel.
- Apply an electric current. Fragments migrate through the gel toward the positive electrode.
- Visualise the bands by staining the gel directly or by transferring to a membrane, probing with labelled DNA probes, and detecting by autoradiography or fluorescence; compare band positions against a DNA ladder.
Two distinct features earn separate marks. Mass — the size, length, or number of nucleotides — determines how fast a fragment moves through the gel mesh; smaller fragments move further. Charge — the negative charge of the phosphate groups in the DNA backbone — is what pulls the fragments toward the positive electrode. Both features are required.
Both features are required. AQA rejects
size alone,density, and any single-feature answer; mass and charge are graded as two separate marks. DNA is always negatively charged; AQA rejectspositive chargefor DNA in gel electrophoresis. The positive electrode is what DNA migrates toward, not the charge it carries.
Applications include forensic identification, paternity testing, medical diagnosis of certain inherited conditions, and breeding programmes in agriculture and conservation. The pattern of bands in a sample lane is the genetic fingerprint; comparing band positions between two samples establishes whether they share the same VNTR pattern.
Fragment count depends on DNA topology. Circular DNA cut at n recognition sites produces n fragments. Linear DNA cut at n sites produces n + 1 fragments. Mis-applying the rule produces a gel diagram with the wrong number of bands.
Pitfall — Order the procedure, don't just name the components
Order the procedure, don't just name the components.
When a question asks for the procedure to screen a DNA sample for a specific allele, the credited answer is the ordered sequence: PCR amplification → restriction endonuclease digest → gel electrophoresis → labelled DNA probe addition → detection by autoradiography or fluorescence → comparison to a known reference banding pattern.
Recalling the components without ordering them caps the answer at half the available marks. The order is the mark.
Gene therapy delivers a functional copy of a faulty gene, with contested benefits.
Gene therapy is the clinical use of recombinant DNA technology to treat a disease caused by a faulty gene. The functional version of the gene is delivered into the patient's cells, where it is expressed and produces the missing or correct protein. Two distinctions structure the spec-level treatment.
The two distinctions that structure spec-level gene therapy.
| Distinction | What it means | Spec-level treatment |
|---|---|---|
| Somatic vs germline | Somatic targets non-reproductive cells (modification not inherited); germline targets reproductive cells (heritable) | AQA answers concern somatic-cell delivery; germline is not in clinical use |
| In-vivo vs ex-vivo | In-vivo introduces the gene directly to the patient; ex-vivo removes cells, modifies them outside the body, reintroduces them | In-vivo example: viral vector to lung tissue for cystic fibrosis. Ex-vivo example: bone marrow stem cells for SCID |
- State at least one For point. For example: no donor required when the patient's own cells are used (ex-vivo); no bone marrow destruction needed under some protocols.
- State at least one Against point. For example: immune response to the viral vector; continued production of faulty cells from untreated stem cells, so the disease is managed but not cured.
- State a conclusion that follows from the points raised.
A one-sided Evaluate-gene-therapy answer is capped at 2 marks. The top mark unlocks only when both For and Against are present. AQA rejects
only own cells usedas equivalent tono donor required; the second phrasing is the credited one. When modified stem cells repopulate bone marrow, the cell division ismitosis; AQA rejectsmeiosis, because gametes are not the targets of somatic-cell gene therapy.
Key terms
- restriction endonuclease
- ligase
- plasmid
- reverse transcriptase
- pcr
- dna polymerase
- dna probe
- gel electrophoresis
- hybridisation
- phosphodiester bond