Principles of genetic engineering

1. Overview

Genetic engineering is the deliberate transfer of a gene from one organism into another so that the gene is expressed and gives the recipient a new, useful characteristic (for example, getting bacteria to make human insulin). The product is recombinant DNA - DNA built from two different sources. To do this, scientists must first obtain the gene, cut and join DNA using specific enzymes, deliver the gene using a vector such as a plasmid, and check it works.

This topic also covers gene editing and the everyday laboratory tools used to copy, separate and analyse DNA:

• the polymerase chain reaction (PCR) - to copy DNA,
• gel electrophoresis - to separate DNA fragments by length,
• microarrays - to analyse genomes and gene expression, and
• sequence databases - to store and share genetic information.

Key Definitions

• Recombinant DNA: DNA made by joining together sections of DNA from two different organisms (or two different sources).
• Genetic engineering: the deliberate manipulation of genetic material to modify the characteristics of an organism, often by transferring a gene so that it is expressed.
• Restriction endonuclease: an enzyme that cuts DNA at a specific base sequence (a recognition site), often leaving short single-stranded sticky ends.
• DNA ligase: an enzyme that joins DNA fragments together by forming phosphodiester bonds in the sugar-phosphate backbone.
• Plasmid: a small circular piece of DNA found in bacteria, separate from the main chromosome, used as a vector to carry a gene into a host cell.
• Reverse transcriptase: an enzyme that uses an mRNA molecule as a template to synthesise a complementary DNA strand (cDNA).
• Promoter: a base sequence positioned before a gene that allows RNA polymerase to bind and start transcription, so the gene can be expressed.
• Marker gene: a gene transferred alongside the desired gene whose product (such as a fluorescent protein) shows whether the host cell has taken up and expressed the new DNA.
• Gene editing: a form of genetic engineering that inserts, deletes or replaces DNA at a specific chosen site in an organism's genome.
• Polymerase chain reaction (PCR): a laboratory technique that rapidly copies (amplifies) a specific section of DNA through repeated cycles of heating and cooling.
• Gel electrophoresis: a technique that separates DNA fragments according to their length by moving them through a gel using an electric field.

Content

What genetic engineering is, and recombinant DNA

Genetic engineering is the deliberate manipulation of genetic material to modify the characteristics of an organism. Most commonly this means transferring a gene from one organism into another so that the gene is expressed - that is, transcribed and translated into a functional protein in the recipient. Because the genetic code is universal (the same triplets code for the same amino acids in nearly all organisms), a gene from one species will normally be read correctly by another.

When DNA from two different organisms is joined together, the result is recombinant DNA. An organism that has received DNA from a different species is described as transgenic.

Three ways to obtain the gene

The desired gene can be obtained in three ways:

• Extracted directly from the donor's DNA - restriction endonucleases are used to cut the gene out of the donor organism's chromosomal DNA.
• Synthesised from mRNA - the mature mRNA for the desired protein is isolated from cells that actively make it. The enzyme reverse transcriptase then uses this mRNA as a template to build a complementary DNA strand, producing cDNA. A key advantage is that cDNA is made from mature mRNA, so it lacks introns - it is the coding sequence only, which matters because bacteria cannot remove introns.
• Synthesised chemically - if the base sequence is known (for example from a database), the gene can be built nucleotide by nucleotide in an automated machine.

The enzymes and vector used to transfer a gene

Several molecules work together to insert a gene into a host. The table below summarises the role of each named molecule - learn this for the "explain the roles of..." objective.

Molecule	Role in transferring a gene
Restriction endonuclease	Cuts DNA at a specific recognition sequence, often leaving sticky ends (short single-stranded overhangs). The same enzyme cuts both the gene and the plasmid, so they get complementary sticky ends.
Plasmid	Acts as the vector - a small circular DNA molecule that carries the gene into the bacterial host cell.
DNA ligase	Seals the gene into the plasmid by forming phosphodiester bonds in the sugar-phosphate backbone, producing a complete recombinant plasmid.
DNA polymerase	Synthesises new DNA strands from a template (e.g. completing or copying a strand), building in the $5' \to 3'$ direction.
Reverse transcriptase	Uses mRNA as a template to make a cDNA copy of the gene.

How the sticky ends fit together: because the same restriction enzyme cuts both the gene and the plasmid, the exposed bases on each are complementary and base-pair with one another. DNA ligase then seals the joins. The recombinant plasmid is taken up by host bacteria, which are cultured so that the gene is expressed.

Why a promoter must also be transferred

A gene is only useful if the host can actually express it. Transcription only starts when RNA polymerase binds to a promoter - a control sequence positioned immediately before the gene. If the donor gene is inserted without a promoter that the host recognises, RNA polymerase cannot bind and the gene stays switched off. So a suitable promoter is transferred along with the gene to ensure it is transcribed and translated in the new organism.

Confirming the gene works using marker genes

After transfer, only some host cells take up the recombinant DNA. A marker gene is included alongside the desired gene to identify which cells have succeeded. Modern markers code for fluorescent products, such as green fluorescent protein (GFP). Cells that have taken up and expressed the recombinant DNA will fluoresce under suitable light, while unsuccessful cells do not. The marker therefore confirms two things at once: that the DNA was taken up, and that it is being expressed. Fluorescent markers have largely replaced the older antibiotic-resistance gene markers - where successful cells survive on a medium containing the antibiotic - because fluorescence reports actual gene expression (not just uptake) and avoids using antibiotic-resistance genes.

Note that this does not change which genes a cell contains. Every body (somatic) cell of an organism carries the full set of genes; what differs between cell types is only which genes are expressed. A marker gene works by reporting on expression in the host cell, not by giving the cell a different gene set from its neighbours.

Gene editing

Gene editing is a form of genetic engineering that does not just add a gene at a random position - it makes a precise change at a specific chosen site in the genome. The change can be:

• insertion of new bases,
• deletion of bases, or
• replacement of one sequence with another.

This precision means a faulty sequence can be corrected exactly where it lies. A clear example is a condition caused by an abnormally long run of repeated bases: gene editing can be directed to delete the extra repeated triplets from the faulty allele while leaving the rest of the genome untouched. When you answer such a question, name the specific change being made rather than just saying "a precise change".

The polymerase chain reaction (PCR)

PCR makes millions of copies of a specific DNA section in a few hours. Each cycle has three temperature stages:

1. Denaturation (~95 °C) - heat breaks the hydrogen bonds between the two strands, separating the DNA into single strands.
2. Annealing (~55 °C) - the mixture is cooled so that short single-stranded primers base-pair to the start of each target sequence, marking where copying begins.
3. Extension (~72 °C) - Taq polymerase adds free DNA nucleotides to each primer, building new complementary strands.

The key to PCR is Taq polymerase, a DNA polymerase from a bacterium that lives in hot springs. It is thermostable, so it is not denatured by the 95 °C denaturation step and does not need replacing each cycle. Because every new strand becomes a template in the next cycle, the amount of DNA doubles each cycle. This doubling gives the only quantitative relationship in the topic - the $2^n$ formula in the Key Equations section below. In practice the $2^n$ doubling is an idealised maximum: as reagents and primers run low the reaction plateaus, so a data question may show the copy number levelling off rather than doubling indefinitely.

Gel electrophoresis

Gel electrophoresis separates DNA fragments by length. DNA samples are placed in wells at one end of an agarose gel, and an electric field is applied. Because DNA is negatively charged (due to its phosphate groups), the fragments move towards the positive electrode (anode). The gel acts as a molecular sieve: shorter fragments move faster and travel further, while longer fragments are held back and move less far. After a set time the fragments form bands, sorted by size, which can be visualised with a stain or label.

Microarrays

A microarray is a slide carrying thousands of tiny spots, each containing a known single-stranded DNA probe. Labelled sample DNA or cDNA is washed over it and binds (hybridises) only where it is complementary to a probe, producing a detectable (often fluorescent) signal. Microarrays are used in two distinct ways:

• Genome analysis - to detect which specific genes or sequences are present in a sample of DNA (for example, screening for particular alleles).
• Gene expression studies - by extracting mRNA, converting it to labelled cDNA, and applying it to the array, the spots that light up reveal which genes were being actively transcribed.

Sequence and structure databases

Vast amounts of biological data are stored in searchable databases holding nucleotide sequences of genes and genomes, amino acid sequences of proteins, and protein structures. Their benefits include: rapidly comparing sequences between species to study evolutionary relationships; identifying genes and predicting the proteins they encode; designing PCR primers and synthetic genes; and sharing data worldwide so researchers can build on each other's work instead of repeating it.

Worked example

Exam-style question: A scientist wants to transfer a human gene into bacteria so that the bacteria produce a human protein. The gene is cut from a plasmid and inserted into another plasmid. Explain why the same restriction endonuclease is used to cut both the gene and the plasmid, and why a marker gene coding for a fluorescent protein is also included. [3]

Model answer:

• Using the same restriction endonuclease on both means the gene and the plasmid are cut to leave complementary sticky ends, so the gene's bases can base-pair with the plasmid's bases.
• This allows DNA ligase to join them, forming a single recombinant plasmid.
• The fluorescent marker gene lets the scientist identify host cells that have taken up and expressed the recombinant DNA, because only those cells fluoresce.

Worked example

Exam-style question: A reaction mixture contains 50 copies of a target DNA sequence. PCR is run for 8 complete cycles. Assuming the amount of target DNA doubles each cycle, calculate the approximate number of copies present after 8 cycles. Then state how many further cycles would be needed to reach at least 1 million copies in total. [3]

Model answer:

• After $n$ cycles the number of copies is the starting number multiplied by $2^n$, so copies $= 50 \times 2^{8}$.
• $2^{8} = 256$, so copies $= 50 \times 256 = 12\,800$ copies.
• To reach $1\,000\,000$ copies, solve $50 \times 2^{n} \ge 1\,000\,000$, i.e. $2^{n} \ge 20\,000$. Since $2^{14} = 16\,384$ and $2^{15} = 32\,768$, you need $n = 15$ cycles in total, which is 7 more than the 8 already run.

Worked example

Exam-style question: A gel is run with four lanes. Lane 1 is a size ladder with bands at $1000$, $500$ and $250$ base pairs (bp), the $1000$ bp band sitting nearest the wells and the $250$ bp band having travelled furthest. Lanes 2, 3 and 4 each contain one DNA fragment: the fragment in lane 2 has travelled further than any ladder band; lane 3 lines up level with the $500$ bp band; lane 4 has barely left the well. State which sample fragment is the longest and which is the shortest, and estimate the size of the fragment in lane 3. [3]

Model answer:

• Shorter fragments travel further, so the lane 2 fragment, which has migrated the furthest of all, is the shortest.
• The lane 4 fragment has moved least, so it is the longest.
• The lane 3 band is level with the $500$ bp ladder band, so its fragment is approximately $500$ bp long.

Key Equations

This topic is mainly qualitative. The one useful quantitative relationship is the amount of DNA produced by PCR. Starting from a known number of target molecules, after $n$ complete cycles the number of copies is approximately $$\text{copies} = (\text{starting copies}) \times 2^{n}$$ because the quantity of DNA doubles with each cycle. If you begin with a single target molecule this simplifies to $\text{copies} = 2^{n}$.

Common Mistakes to Avoid

• Saying mRNA is "converted" or "changed" into cDNA. State precisely that the mRNA acts as a template and reverse transcriptase synthesises a complementary DNA (cDNA) strand from it.
• Using a "different" restriction enzyme for the gene and the vector. The same restriction endonuclease must cut both, so that the sticky ends are complementary and can base-pair; different enzymes would give ends that do not match.
• Forgetting the promoter. A transferred gene will not be expressed unless a promoter that the host recognises is also transferred, so RNA polymerase can bind and start transcription.
• Saying a marker gene only shows the cell took up the DNA. A fluorescent marker confirms the cell has both taken up and expressed the recombinant DNA - expression is needed for the cell to fluoresce.
• Giving a generic answer for a named gene-editing application. When a question is about a specific condition, say exactly what the edit does - for example, deleting the extra repeated triplets from the faulty allele - rather than only writing "a precise change at a specific site". Vague answers lose the application marks.
• Thinking different cell types contain different genes. All body (somatic) cells of an organism carry the same full set of genes; only the genes that are expressed differ. Do not say a specialised cell "lacks" the genes it is not using.
• Mixing up the two uses of microarrays. Detecting specific DNA sequences is genome analysis; detecting mRNA (as cDNA) is a study of gene expression - read the question and use the matching one.
• Describing PCR vaguely as "heating DNA". Name and explain all three stages with their roles: denaturation (~95 °C), annealing of primers (~55 °C) and extension by Taq polymerase (~72 °C).
• Saying ordinary DNA polymerase is used in PCR. Taq polymerase is needed because it is thermostable and is not denatured by the repeated 95 °C heating.
• Getting the direction of migration wrong in electrophoresis. DNA is negatively charged, so it moves to the positive electrode (anode), and shorter fragments travel further, not the reverse.
• Giving a vague definition of gene editing. Make clear it is precise - the insertion, deletion or replacement of DNA at a specific site in the genome.

Exam Tips

• When asked for the role of an enzyme, name the enzyme and what it does in one sentence (e.g. "DNA ligase joins the gene to the plasmid by forming phosphodiester bonds"); a list of enzyme names alone earns little.
• For PCR questions, link each temperature to its purpose (denaturation, annealing, extension) - credit comes from giving the temperature and the reason, not just the number.
• For PCR calculations, use $\text{copies} = (\text{starting copies}) \times 2^{n}$, and show the power of 2 you used - working backwards to find $n$ is a common twist.
• Always explain why cDNA is useful: it is made from mature mRNA so it has no introns, which matters because bacteria cannot splice introns out.
• Use the precise term complementary for sticky ends and for probe binding, rather than "the same" or "matching".
• In electrophoresis answers, state the charge on DNA, the direction it moves, and that length determines how far each fragment travels - give all three for full marks.
• When describing how marker genes confirm success, stress that fluorescence shows the gene was both taken up and expressed.
• Match your depth to the command word: microarrays and databases are "outline" topics, so give a brief what-it-does-and-why answer, whereas PCR, electrophoresis and enzyme roles are "describe and explain", needing the mechanism and the reason.
• Keep "genetic engineering", "gene editing" and "gene therapy" distinct: editing means a precise change at a specific site; transferring a gene with a vector is the broader engineering process.