Recombinant DNA technology — the seven-step pipeline
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define the term recombinant DNA technology. Describe and explain the stages of recombinant DNA technology, limited to: isolation of the target gene, insertion of the target gene into a vector, introduction of the vector into a host organism, selection of the host organism that has taken up the gene, replication of the gene and translation of the gene to produce the recombinant protein.
Why "recombinant"?
Recombinant DNA is any DNA molecule that has been made by joining together DNA from two different sources. The word recombinant simply means "recombined" — pieces of DNA that did not exist together in nature have been combined in the test tube. The 2025 M/J /42 Q5(c) is a typical 1-mark definition question: "explain why the DNA involved in the production of recombinant human insulin is termed recombinant DNA". The MS gives the mark for stating that the DNA is made by joining together (DNA from) two different sources / species / organisms — for insulin, the human insulin gene joined to a yeast or bacterial plasmid.
Recombinant DNA technology is the set of techniques used to make a recombinant DNA molecule, get it into a living cell, and have the cell read the new gene and make the protein. The MS in 2024 O/N /42 Q3(a) gives the standard definition: the deliberate modification of the genome of an organism by the transfer of a gene from one organism to another, so that the recipient organism produces the gene product.
The point of the technology is production — once the gene is in the host cell, every time the host divides it copies the new gene, and the host's ribosomes read the new gene and make the protein. A fermenter of yeast making human insulin is a more efficient source of insulin than extracting it from the pancreases of pigs and cattle.
Fig 19.1 The seven-step pipeline of recombinant DNA technology. From isolating the gene in a donor cell to expressing the new protein or trait in a host organism. Steps 1–4 happen in the test tube; step 5 puts the recombinant plasmid into a living host; step 6 is selection (only the transformed cells survive); step 7 is expression of the new protein.
The seven steps, one at a time
Step 1 — isolate the gene. The first job is to get a copy of the gene you want. §02 covers the three named methods: isolating the gene from a donor organism's DNA, copying it from mRNA using reverse transcriptase, and synthesising it from nucleotides.
Step 2 — cut the plasmid with a restriction enzyme. Restriction enzymes (see §03) recognise a specific short sequence of bases (a recognition sequence or restriction site) and cut the DNA at that site, leaving either blunt ends or short single-stranded overhangs (sticky ends). Cutting the plasmid with the same enzyme as the gene leaves matching sticky ends, so the gene can slot in.
Step 3 — ligate the gene into the plasmid. DNA ligase (see §03) joins the sugar–phosphate backbones of the gene and the plasmid, sealing the recombinant plasmid. The result is a small circular DNA molecule that contains the new gene, a promoter (so the gene can be transcribed), a marker gene (so transformed cells can be selected — see §04), and an origin of replication so the plasmid can be copied inside the host cell.
Step 4 — add promoter and marker gene to the plasmid. In practice the promoter and marker gene are already carried on the plasmid before the gene is inserted. The plasmid is engineered to carry a multiple-cloning site (a short sequence with restriction sites for several different enzymes), a strong promoter upstream of that site, and a marker gene (often an antibiotic-resistance gene or a fluorescent gene such as GFP). The student is expected to know what each of these does and why each is needed (see §04).
Step 5 — introduce the recombinant plasmid into a host cell. This is called transformation in bacteria and yeast, and transfection in animal cells. The most common methods are heat-shock (a brief 42 °C pulse that makes the bacterial membrane permeable to the plasmid), electroporation (a brief electric pulse that opens transient pores in the membrane), and viral delivery (for animal cells — see §10). Not every cell takes up a plasmid; the next step selects the ones that did.
Step 6 — select the cells that have taken up the plasmid. The marker gene is the trick here. If the marker is an antibiotic-resistance gene, the transformed cells are grown on a medium containing the antibiotic; cells without the plasmid are killed, cells with the plasmid survive. If the marker is a fluorescent gene, the transformed cells glow under UV light and can be sorted by a fluorescence-activated cell sorter (FACS). §04 covers the marker-gene mechanism in detail, including insertional inactivation — placing the gene of interest inside the marker gene so that successful insertion switches off the marker, giving a second layer of selection.
Step 7 — replicate and express. The transformed cells are grown in a fermenter on an industrial scale. As the cells divide, the plasmid (and the gene it carries) is copied, and the cell's ribosomes read the gene and make the protein. For insulin, the protein is harvested from the culture medium and purified. For a GM crop, the seed of the transformed plant is harvested and the gene (and the trait it confers) is inherited by the next generation.
Why this pipeline matters in the exam
Almost every "outline the process" question on §19.1 expects the student to name the seven steps in the right order and to give the role of each tool or feature. The 2024 O/N /42 Q3(a) asked for a definition of genetic engineering (3 marks); the 2025 M/J /42 Q5(a) asked for a description of producing recombinant human proteins (3 marks); the 2025 O/N /42 Q5(a) was on the reagents required for PCR (4 marks — covered in §06). Across the 2021–2025 window, ~30 marks a year go to "outline the steps" or "explain the process" questions on this topic. The safe seven steps, in order, are: (1) isolate / obtain the gene; (2) cut the plasmid and the gene with the same restriction enzyme; (3) ligate the gene into the plasmid; (4) introduce the recombinant plasmid into a host cell; (5) select the cells that have taken up the plasmid (marker gene); (6) allow the cells to divide and copy the gene; (7) harvest the protein.
Explaining why the DNA in recombinant human insulin is 'recombinant' (a 1-mark definition)
A number of diseases in humans can be treated using recombinant human proteins. These are produced by recombinant DNA technology.
Explain why the DNA involved in the production of recombinant human insulin is termed recombinant DNA.
Show full working
- 1
The DNA is made by joining together (DNA from) two different sources / species / organisms. The recombinant insulin DNA is a hybrid — the human insulin gene joined to a yeast (or bacterial) plasmid. The "two different sources" framing is what the MS awards the mark for; the MS rejects the looser "the DNA has been changed" answer.
Mark 1. The MS gives the mark for the join of DNA from two different sources.
The DNA is termed recombinant because it is made by joining together DNA from two different sources / species — a human gene (the insulin gene) joined to a plasmid (from yeast or bacteria).
On a 1-mark definition of recombinant DNA, the safe phrasing is 'DNA made by joining together DNA from two different sources / species / organisms'. The MS credits the join of two sources; the MS rejects the looser 'the DNA has been changed' answer because that does not specify the two-source structure.
Your turn — the recombinant DNA pipeline
- 13 marks
Explain what is meant by genetic engineering.
Stuck? Show hint
Three marks: (1) deliberate / intentional modification of the genome; (2) by the transfer of a gene (or allele) from one organism to another; (3) so that the recipient organism produces the gene product.
Show solution
- 1
Deliberate modification of the genome. Genetic engineering is the intentional change to the genome of an organism. The MS credits the word "deliberate" (or a synonym such as "intentional" / "planned") because the definition has to rule out natural mutation.
Mark 1. The MS credits the deliberate modification.
- 2
By the transfer of a gene / allele from one organism to another. A gene (or allele) is moved from a donor organism to a recipient organism. The MS uses "one organism to another" — the recipient and donor do not have to be from different species, but the two-source structure is what makes the DNA recombinant.
Mark 2. The MS credits the transfer of a gene.
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So that the recipient organism produces the gene product. The point of the technology is expression — the recipient cell reads the new gene and makes the protein. The MS credits the outcome clause.
Mark 3. The MS credits the outcome.
AnswerGenetic engineering is the deliberate modification of the genome of an organism by the transfer of a gene (or allele) from one organism to another, so that the recipient organism produces the gene product.
- 1
- 23 marks
Place the seven steps of recombinant DNA technology in the correct order:
- A. add promoter and marker gene to the plasmid
- B. select the host cells that have taken up the plasmid
- C. isolate the target gene from the donor organism
- D. cut the plasmid and the target gene with a restriction enzyme
- E. ligate the target gene into the plasmid
- F. introduce the recombinant plasmid into a host cell
- G. harvest the recombinant protein
Stuck? Show hint
The correct order is C → D → E → A → F → B → G.
Show solution
- 1
First two steps: C (isolate the gene) and D (cut with restriction enzyme). The gene is isolated from the donor; the plasmid and the gene are cut with the same restriction enzyme so that they share the same sticky ends.
Mark 1. The MS credits C → D in the correct order at the start.
- 2
Middle steps: E (ligate) and A (add promoter and marker). DNA ligase joins the gene into the plasmid; in practice the promoter and marker gene are already on the plasmid, but the gene must be inserted in the right place — downstream of the promoter.
Mark 2. The MS credits E → A in the correct order in the middle.
- 3
Last three steps: F (introduce into host) and B (select) and G (harvest). The recombinant plasmid is introduced into the host cell; the host cells that have taken up the plasmid are selected using the marker gene; the protein is harvested from the culture.
Mark 3. The MS credits F → B → G in the correct order at the end.
AnswerC → D → E → A → F → B → G. (isolate the gene → cut with restriction enzyme → ligate into plasmid → add promoter and marker gene → introduce into host cell → select transformed cells → harvest the protein.)
The rest of this note
Can you do all of these?
Define recombinant DNA as DNA made by joining together DNA from two different sources / species / organisms
Define recombinant DNA technology as the deliberate modification of the genome of an organism by the transfer of a gene from one organism to another, so that the recipient organism produces the gene product
Name the seven steps of recombinant DNA technology in the correct order: (1) isolate the gene; (2) cut plasmid and gene with the same restriction enzyme; (3) ligate the gene into the plasmid; (4) add promoter and marker gene; (5) introduce into host cell; (6) select transformed cells; (7) harvest the protein
Name the three sources of a target gene: (1) isolation from a donor organism; (2) cDNA from mRNA via reverse transcriptase; (3) chemical synthesis from nucleotides
Explain why cDNA (not genomic DNA) is preferred for expression in a bacterial or yeast host — cDNA has no introns, and bacteria have no spliceosome
Describe the role of a restriction enzyme: cuts DNA at a specific recognition sequence, leaving short single-stranded sticky ends
Describe the role of DNA ligase: joins the sugar–phosphate backbones of two DNA fragments, sealing the recombinant plasmid
Describe the role of a plasmid: a small circular DNA molecule used as a vector to carry the new gene into the host cell and to replicate inside it
Describe the role of DNA polymerase: synthesises a new DNA strand using a DNA template (PCR extension; cDNA second strand)
Describe the role of Taq polymerase: a thermostable DNA polymerase from Thermus aquaticus that survives the 95 °C denaturation step of every PCR cycle
Describe the role of reverse transcriptase: synthesises a cDNA copy of an mRNA template
Describe the role of a promoter: a DNA sequence upstream of the gene that binds RNA polymerase, allowing the gene to be transcribed
Describe the role of a marker gene: a gene that gives the transformed cell a selectable phenotype (antibiotic resistance or fluorescence) so that the transformed cells can be selected
Describe insertional inactivation: placing the gene of interest inside the marker gene so that successful insertion disrupts the marker; cells with the recombinant plasmid are identified by their loss of the marker phenotype
Outline the principles of CRISPR/Cas9: a guide RNA complementary to the target binds to the target by complementary base-pairing, a Cas9 nuclease cuts the DNA at the target, the cell's repair mechanisms repair the cut, with the possibility of a template DNA being inserted
Distinguish gene editing (correct the mutation in situ) from gene addition therapy (give a new copy of the gene)
Describe PCR: a method for making many copies of a specific DNA sequence from a small starting amount; the three steps are denaturation (95 °C), annealing (~55 °C) and extension (72 °C); after n cycles, ~2ⁿ copies
Describe gel electrophoresis: DNA is negatively charged (phosphate groups), so it migrates toward the anode (positive electrode); smaller fragments move further through the gel matrix than larger ones; the DNA ladder is a set of fragments of known size used to estimate the size of sample bands
Outline how microarrays work: thousands of gene-specific probes are fixed to a slide; fluorescent cDNA is washed over the slide; a glowing spot = the gene is expressed in the sample
Outline how a DNA sequence database is used: the unknown sequence is entered into BLAST (or similar); the tool finds regions of similarity; the closest match identifies the unknown sequence
Describe the production of recombinant human insulin: mRNA from human pancreatic β-cells → cDNA via reverse transcriptase → cDNA inserted into a plasmid with a promoter and a marker gene → plasmid introduced into a yeast (or E. coli) host → transformed cells selected and grown in a fermenter → insulin harvested and purified
Give two advantages of recombinant human insulin over animal-derived insulin: no risk of pathogen transmission; identical amino-acid sequence to human insulin / unlimited supply
Outline the principles of genetic screening for BRCA1, BRCA2, Huntington's and cystic fibrosis: DNA sequencing (often after PCR amplification); identifies the specific mutation; the result is a diagnosis, a risk assessment or a prognosis
Outline the principles of gene therapy: a virus (usually AAV for in-vivo, lentivirus for ex-vivo) is used to deliver a functional copy of the gene into the patient's cells; the gene is integrated into the cell's genome and is expressed to produce a functional protein
Distinguish ex-vivo (cells taken out, modified, returned) from in-vivo (vector injected directly into the patient) gene therapy; the named conditions are SCID (ex-vivo) and LCA2 (in-vivo)
Discuss the ethical implications of genetic screening and gene therapy: autonomy / informed consent; confidentiality / discrimination; equity of access; reproductive decisions; no-treatment diseases (Huntington's)
Outline the production of herbicide-resistant soybean: a gene for glyphosate resistance from Agrobacterium tumefaciens is inserted into the soybean genome; the farmer can spray the field with glyphosate, killing weeds but not the crop
Outline the production of insect-resistant cotton: a Bacillus thuringiensis (Bt) Cry toxin gene is inserted into the cotton genome; the cotton produces the Bt toxin in its tissues; the Bt toxin kills the cotton bollworm larvae that feed on the cotton
Outline the production of AquAdvantage salmon: a Chinook salmon growth-hormone gene, controlled by an ocean pout antifreeze-protein promoter, is inserted into the Atlantic salmon genome; the salmon expresses the growth-hormone gene all year round and reaches market size in half the time
Discuss the implications of GMOs in food production: higher yield / lower cost; reduced pesticide use; reduced soil damage; concerns about human health (allergenicity / toxicity); concerns about the environment (gene transfer to wild relatives; superweeds; harm to non-target insects; escaped GM salmon); concerns about corporate control; concerns about labelling and consumer choice