Recombinant DNA technology involves transferring fragments of DNA from one organism, or species, to another. Because the genetic code is universal, the transferred DNA can be translated in the recipient. Fragments of DNA can be produced by conversion of mRNA to complementary DNA using reverse transcriptase, by using restriction endonucleases to cut a fragment containing the desired gene, and by creating the gene in a gene machine. DNA fragments can be amplified using in vivo techniques involving vectors and the use of the polymerase chain reaction (PCR) in vitro. The use of recombinant DNA technology to produce transformed organisms that benefit humans, and the use of gene therapy.

What this dot point is asking

AQA wants you to explain the whole pipeline of genetic engineering: why it works at all (universal code), three ways to isolate a gene, two ways to amplify DNA (in vivo with vectors, in vitro with PCR), how to transform a recipient and identify success with marker genes, and the principle of gene therapy. This is a large dot point, so structure your revision around the stages.

Why it works: the universal code

The genetic code is universal: the same triplet codes for the same amino acid in (almost) all organisms. This means a gene from one species can be transcribed and translated correctly when transferred into a completely different species. A human gene can therefore be expressed by a bacterium.

Step 1: isolating the gene (making DNA fragments)

There are three methods in the specification.

Reverse transcriptase.

1. Isolate the mRNA for the gene from a cell that makes a lot of the protein (the mRNA is abundant there).
2. The enzyme reverse transcriptase uses the mRNA as a template to make single-stranded complementary DNA (cDNA).
3. DNA polymerase makes the complementary second strand, producing double-stranded DNA. An advantage is that cDNA contains no introns.

Restriction endonucleases.

1. A restriction endonuclease cuts DNA at a specific base sequence called a recognition site.
2. Cutting on each side of the gene releases the fragment.
3. Many restriction enzymes make a staggered cut, leaving short single-stranded overhangs called sticky ends, which can base-pair with complementary sticky ends on a vector.

The gene machine.

The base sequence of a gene can be looked up from the desired amino acid sequence, and the gene synthesised chemically. This avoids introns and errors and can produce any sequence, including those not found in nature.

Step 2: amplifying the DNA

In vivo (inside a living organism) using vectors.

1. The desired gene and a vector (commonly a bacterial plasmid) are cut with the same restriction endonuclease, so they have complementary sticky ends.
2. The gene and the vector are mixed; DNA ligase joins them by forming phosphodiester bonds, producing recombinant DNA.
3. The recombinant plasmid is taken up by host cells (e.g. bacteria) in transformation. Uptake is encouraged with calcium ions and heat shock.
4. The transformed cells are grown; as they divide, they copy the plasmid, amplifying the gene and (if expressed) making the protein.

Marker genes. Only some cells take up the plasmid. A marker gene (for example antibiotic resistance, fluorescence or an enzyme that changes colour) is included so that transformed cells can be identified and selected.

In vitro (in a test tube) using PCR.

The polymerase chain reaction (PCR) amplifies DNA rapidly without living cells. Each cycle has three temperature stages:

1. Denaturation (about 95 degrees C). Heat breaks the hydrogen bonds, separating the two DNA strands.
2. Annealing (about 55 degrees C). Short single-stranded primers bind to the start of each target strand by complementary base pairing.
3. Extension (about 72 degrees C). A heat-stable DNA polymerase (Taq) extends from each primer, building a new complementary strand.

Each cycle doubles the amount of DNA, so $n$ cycles give a factor of $2^n$ increase.

Step 3: making useful transformed organisms

Transformed organisms benefit humans in many ways, for example:

• Bacteria transformed with the human insulin gene produce human insulin to treat diabetes.
• Other transformed microbes make medicines, enzymes and hormones.
• Genetically modified crops can be made pest-resistant, herbicide-tolerant or more nutritious.

There are ethical, social and economic arguments to weigh, but the biological principle is the same: a useful gene is transferred and expressed.

Gene therapy

Gene therapy aims to treat a genetic disorder by supplying a working version of a faulty gene.

• In a recessive disorder, a working dominant allele is inserted so a functional protein is made.
• The allele is delivered using a vector such as a harmless virus or a liposome.
• Somatic gene therapy targets body cells (e.g. lung cells in cystic fibrosis); it treats the patient but is not inherited.
• Germ-line gene therapy targets gametes or embryos and would be inherited, but it raises serious ethical concerns and is not permitted in humans in many countries.

A practical difficulty is that somatic gene therapy is often temporary, because treated cells die and are replaced by untreated cells, so treatment must be repeated.

Common mistakes

Try this

Q1. Explain one advantage of using cDNA made by reverse transcriptase rather than cutting the gene from genomic DNA. [2 marks]

• Cue. cDNA is made from mature mRNA, so it contains only exons and no introns; this is important because bacteria cannot splice out introns, so the gene can be expressed correctly.

Q2. Calculate the number of DNA molecules produced from one starting molecule after 10 cycles of PCR. [1 mark]

• Cue. $2^{10} = 1024$ molecules.

Q3. Explain why a marker gene is needed when transforming bacteria with a plasmid. [2 marks]

• Cue. Not all bacteria take up the plasmid; the marker gene allows transformed bacteria (which also carry the marker) to be identified and selected from non-transformed cells.