3.8.4.1 - Recombinant DNA technology

Producing DNA fragments

A human gene can work inside a bacterium, but first it has to be obtained as a DNA fragment.

Recombinant DNA technology is the transfer of DNA fragments from one organism, or species, to another. It works because the genetic code is universal: the same codons specify the same amino acids in almost all organisms, and the core mechanisms of transcription and translation are shared.

So the recipient can translate the transferred DNA, and it is described as transgenic. In practice, expression in a different organism also requires suitable regulatory sequences, and in bacterial hosts the coding sequence must not contain introns.

A gene is a tiny part of a genome that can run to billions of base pairs, so you need a precise way to obtain it. There are three main routes.

Reverse transcriptase and cDNA

A beta cell in the pancreas has two copies of the insulin gene but holds a large amount of insulin mRNA. So you can start from that plentiful mRNA and copy it back into DNA, using the enzyme you met in HIV.

The diagram's four panels match the four steps below it.

[DIAGRAM: asset_name: 3.8.4.1-cdna-v2; asset_slug: 3.8.4.1-cdna-v2; recommended_method: retained_png; caption: Mature mRNA is copied into cDNA, the RNA template is removed, and a second DNA strand is synthesised.; description: Mature mRNA is copied into cDNA, the RNA template is removed, and a second DNA strand is synthesised.]

1. Extract mature mRNA from a cell type that expresses the gene abundantly, such as the beta cells of the islets of Langerhans.
2. Make the first DNA strand. Reverse transcriptase joins free DNA nucleotides that are complementary to the mRNA, giving an RNA–DNA hybrid. The new strand is complementary DNA (cDNA).
3. Remove the RNA template, leaving single-stranded cDNA.
4. Make the second strand. DNA polymerase adds nucleotides complementary to the cDNA template, producing a double-stranded DNA copy of the gene.

Mature mRNA has already been spliced, so its cDNA has no introns. This matters because prokaryotic cells lack the machinery to remove introns: a bacterium can use the intron-free coding sequence when suitable control sequences are supplied. Eukaryotic proteins that require particular processing or modifications may still need a different host.

Restriction endonucleases

The second route cuts a fragment containing the gene directly out of the organism's DNA.

Each enzyme's active site is complementary to a specific short base sequence, so different enzymes cut at different sites and cuts are not random.

At its site, the enzyme breaks phosphodiester bonds in both DNA strands. To remove a gene, use an enzyme whose recognition sites lie either side of it. The fragment keeps any introns the gene has.

The way the enzyme cuts decides what you can do with the fragment:

• Blunt ends: the enzyme cuts straight across both strands at the same position. HpaI recognises GTTAAC and cuts between the T and A on both strands.
• Sticky ends: the enzyme makes a staggered cut, leaving short single-stranded overhangs of unpaired bases. HindIII recognises AAGCTT and cuts between the two A bases on each strand. AAGCTT reads the same 5′ to 3′ on both strands, so the two overhangs are complementary.

An enzyme recognises the same short sequence wherever it occurs, so it leaves matching sticky ends on DNA from any organism. This is what allows fragments from different species to be joined, the basis of recombinant DNA.

Compatible sticky ends make it easier to align an insert with a vector. Blunt-ended DNA can also be joined, but it lacks this initial pairing.

The gene machine

The third route needs no DNA or RNA template, because the gene is built from its sequence.

1. If the protein's amino acid sequence is known, scientists use the genetic code to design a possible coding DNA sequence. The code is degenerate, so more than one DNA sequence may code for the protein: the sequence is designed, not uniquely read off.
2. A computer designs a series of short, overlapping single-stranded sequences called oligonucleotides.
3. An automated synthesiser assembles each oligonucleotide one nucleotide at a time.
4. The oligonucleotides are joined to form the gene, the complementary strand is synthesised, and the finished gene can be copied by PCR (next section).

A chosen sequence can be produced rapidly and with high accuracy, designed without introns and with codons suitable for the host. Expression still requires compatible regulatory sequences: synthesising the DNA alone does not produce protein.

Amplifying DNA fragments

One fragment is far too little DNA to work with, so it is copied, either in a tube or inside living cells.

The tube method, PCR, is in vitro: outside living cells. The other method is in vivo: the culture of host cells that have taken up the DNA, called transformed cells (see transformation, in the next section).

The polymerase chain reaction (PCR)

PCR is carried out in a thermocycler, a programmable machine that precisely controls temperature. The reaction mixture contains:

• the target DNA fragment
• free DNA nucleotides
• two short primers, single-stranded sequences complementary to the ends of the target fragment
• a heat-stable DNA polymerase called Taq polymerase, from the hot-spring bacterium Thermus aquaticus

The principle is that each strand made in one cycle becomes a template in the next. Each cycle has three temperature-controlled steps, shown left to right in the diagram.

[DIAGRAM: asset_name: 8.4.1 - Recombinant DNA technology - Diagram 2; asset_slug: 8.4.1 - Recombinant DNA technology - Diagram 2; recommended_method: retained_png; caption: One PCR cycle separates strands, binds primers and extends them, repeated ideal cycles double the DNA copies.; description: One PCR cycle separates strands, binds primers and extends them; repeated ideal cycles double the DNA copies.]

1. Denaturation (95 °C). The high temperature breaks the hydrogen bonds between complementary base pairs, separating the DNA into two single strands.
2. Annealing (often around 50–65 °C). Cooling lets the primers bind (anneal) to complementary sequences at each end of the target. Primers are in excess, which favours this binding, although strands can also re-anneal. DNA polymerase can only add nucleotides to the end of an existing strand, so primers provide the starting point. Synthesis begins only where a primer has bound, so only the DNA lying between the two primers is amplified.
3. Elongation (about 72 °C for Taq). This is close to Taq's optimum temperature. Taq extends each primer by adding complementary nucleotides along the template strand, giving two double-stranded copies of the target.

Taq is thermostable, so it survives the 95 °C steps and the same enzyme can work through later cycles. Annealing temperature depends on the primers; the diagram's 55 °C is one possible value.

The lower row of the diagram shows the result: in the idealised model each cycle doubles the number of target molecules, so one becomes two, then four.

Use this model when a question states perfect doubling. Actual reactions become less efficient as reagents are used up.

In vivo amplification, compared with PCR

The alternative is to clone the DNA inside living cells. The fragment is inserted into a vector, a carrier such as a plasmid (the next section shows how). The vector is introduced into bacterial host cells, which are cultured.

As these transformed cells grow and divide, the vector replicates, and the inserted DNA with it, giving many copies of the gene. PCR is often faster for obtaining DNA. The advantage of culture is that it maintains a clone and can make the gene's protein product.

Neither method is automatically error-free: polymerase choice, culture conditions and sequence checking all matter. Contamination troubles PCR when the contaminating DNA contains the target region recognised by the primers. PCR does not amplify every contaminating sequence.

Cover the table, then explain from memory how the two methods differ.

Inserting DNA and transforming hosts

A gene needs control regions and a carrier before a host cell will copy it and express it.

For in vivo cloning, the carrier is a vector. The most commonly used vectors are plasmids: small, circular DNA molecules found naturally in bacteria, separate from the main bacterial chromosome.

Preparing the fragment with control regions

A coding sequence alone is not enough, because transcription needs a start and a stop. A promoter is not transcribed, so a cDNA copy of mature mRNA has none. Suitable regulatory sequences must be provided, either added to the fragment or already positioned in an expression vector:

• A promoter region is a DNA sequence upstream of the gene. RNA polymerase, with transcription factors in eukaryotes, binds here to initiate transcription.
• A terminator region is a sequence downstream of the gene. It causes RNA polymerase to stop transcription and detach.

The promoter must be recognised by the host machinery and orientated correctly relative to the coding sequence. The terminator ends transcription; it is not the same as a stop codon, which ends translation.

Joining the fragment to the plasmid

Cut a plasmid and a fragment with the same restriction endonuclease and their sticky ends are complementary, so they can pair. A second enzyme then makes the join permanent.

Follow panels 1 to 3 of the diagram. The strip beneath them shows the order of the parts needed for expression: promoter, coding sequence, terminator.

[DIAGRAM: asset_name: 3.8.4.1-recombinant-v2; asset_slug: 3.8.4.1-recombinant-v2; recommended_method: retained_png; caption: Compatible sticky ends base-pair before ligase seals the DNA backbones, expression needs suitable control sequences.; description: Compatible sticky ends base-pair before ligase seals the DNA backbones; expression needs suitable control sequences.]

1. The restriction endonuclease cuts the plasmid at a suitable recognition site; in some designs the site lies within a marker gene.
2. The DNA fragment, with its matching sticky ends, is mixed with the opened plasmids.
3. Complementary sticky ends base-pair through hydrogen bonding.
4. DNA ligase catalyses the formation of phosphodiester bonds, sealing the sugar–phosphate backbone on both strands and permanently joining the fragment into the plasmid.

The result is a recombinant plasmid: a plasmid containing DNA from two different sources.

Transformation

The recombinant plasmids now have to get into the host cells.

The plasmids are mixed with bacterial host cells in a medium containing calcium ions (Ca²⁺). A brief heat shock follows, a rapid temperature shift from around 0 °C to approximately 42 °C. Together these increase the permeability of the bacterial cell-surface membrane, so plasmids can pass into the cytoplasm.

Only a proportion of cells take up a plasmid, depending on the method and conditions. Some of those take up a plasmid with no fragment, because a cut plasmid's two sticky ends are complementary to each other, so it can close back up. Identifying the right cells is therefore the next step.