Biotechnology

PCR: copying one stretch of DNA billions of times

PCR is DNA replication in a test tube, aimed at one region. You mix the DNA you want to copy (the template) with four ingredients: two primers, short single-stranded DNA pieces (typically about 20 bases) that match the two ends of the target region; the four DNA nucleotides (dNTPs); a heat-stable DNA polymerase; and a buffer with Mg²⁺. Then a machine called a thermal cycler heats and cools the tube repeatedly.

1. 1Denaturation (about 94–98 °C): the heat breaks the hydrogen bonds between the strands, separating the double helix into single strands.
2. 2Annealing (about 50–65 °C): as the tube cools, the primers base-pair with their matching sequences at each end of the target. The exact temperature depends on the primers' sequences.
3. 3Extension (about 72 °C): the polymerase adds nucleotides to each primer's 3′ end, building new strands 5′→3′ across the target region.
4. 4Repeat: each cycle roughly doubles the number of copies of the target. After n cycles, one starting molecule can give up to 2ⁿ copies; 30 cycles gives over a billion.

Why primers? Because, as in the cell, DNA polymerase can't start a new strand from nothing. It can only extend an existing 3′ end. The primers give it a starting point, and their positions decide exactly which region gets copied: one primer pairs with each strand, and the two point towards each other.

The polymerase has to survive being heated to about 95 °C every cycle. Early PCR used an ordinary bacterial polymerase that was destroyed by each heating step, so fresh enzyme had to be added every cycle. The fix was Taq polymerase, from Thermus aquaticus, a bacterium first isolated from hot springs in Yellowstone National Park. Its polymerase works happily at 72 °C and survives the heat.

Gel electrophoresis: sorting DNA by size

To see what PCR (or any other step) made, you run the DNA on a gel. Gel electrophoresis uses an electric field to pull DNA through a slab of gel, usually agarose, which is a mesh of tiny pores.

1. 1Load the DNA samples into small wells at one end of the gel.
2. 2Switch on the current. DNA's phosphate backbone is negatively charged, so all DNA moves towards the positive electrode, away from the wells.
3. 3The gel mesh slows larger fragments more than small ones, so small fragments travel farther in the same time.
4. 4Stain the DNA with a dye that glows under the right light. Fragments of the same size form a band.
5. 5Compare with a DNA ladder, a lane of fragments of known sizes, to estimate each band's size.

Every DNA molecule carries about the same charge per unit length, so the push per base is similar for small and large fragments. What separates them is how easily they wriggle through the mesh. The relationship isn't linear: over the gel's useful range, distance travelled falls roughly in a straight line as the logarithm of size rises, so bands of large fragments are squeezed closer together near the wells.

In DNA fingerprinting, PCR copies several regions whose length varies from person to person, such as short repeated sequences. You inherit one version of each region from each parent, so each person has up to two bands per region. Comparing bands can link a suspect to a crime scene sample or test who a child's biological parents are.

Cutting and pasting: restriction enzymes and plasmids

Restriction enzymes are bacterial enzymes that cut DNA at specific short sequences, often 4–8 bases long and palindromic (reading the same 5′→3′ on both strands). Many cut the two strands at slightly different points, leaving short single-stranded overhangs called sticky ends. Any two pieces cut by the same enzyme have matching sticky ends, so they can base-pair with each other.

A plasmid is a small circle of DNA that replicates inside a bacterium separately from its chromosome. Plasmids used as vectors (DNA carriers) are engineered to have a multiple cloning site, a short stretch with many unique restriction sites, and a selectable marker, usually an antibiotic resistance gene.

1. 1Cut both the plasmid and the DNA containing your gene with the same restriction enzyme (or pair of enzymes), giving matching sticky ends.
2. 2Mix them. The sticky ends pair up, and DNA ligase seals the backbone, giving a recombinant plasmid with your gene inserted.
3. 3Put the plasmids into bacteria. Taking up outside DNA like this is transformation. Only a small fraction of cells take up a plasmid.
4. 4Grow the bacteria on a plate with the antibiotic. Only cells carrying a plasmid, with its resistance gene, survive.
5. 5Screen the survivors to find ones whose plasmid actually contains the insert (see below), then grow them up. Each colony is a clone of one cell, so all its cells carry the same plasmid.

Blue–white screening: the lac operon at work

Antibiotic selection tells you a cell has a plasmid, but not whether the plasmid has your insert: some plasmids just close back up empty. Many vectors solve this by putting the multiple cloning site inside a piece of the lacZ gene (lacZα), which in specially engineered host bacteria completes a working β-galactosidase.

• The plates contain X-gal, a colourless compound that β-galactosidase splits, releasing a product that forms a blue dye, and usually IPTG to switch on lac expression.
• Empty plasmid: lacZα is intact, β-galactosidase works, and the colony turns blue.
• Plasmid with insert: the insert disrupts lacZα, no working enzyme is made, and the colony stays white.
• So you pick the white colonies.

IPTG is used rather than lactose because, as you saw in the lac operon lesson, it releases the lac repressor directly, enters without permease and isn't broken down. The same trick is used to switch on genes placed under lac control in expression vectors, which make bacteria produce large amounts of a protein, such as human insulin.

Reading DNA: sequencing

DNA sequencing determines the order of bases. The classic method, developed by Fred Sanger in the 1970s, is chain-termination sequencing. It copies the DNA with a polymerase in the presence of normal nucleotides plus a small amount of dideoxynucleotides (ddNTPs), which lack the 3′-OH group. When a ddNTP is added, nothing can be attached after it, so that copy stops.

The result is a set of copies ending at every possible position. In the modern version, each copy is tagged with a coloured dye for its last base (A, T, G or C). Sorting them by length, one base apart, and reading the colours in order spells out the sequence. It's the 5′→3′, 3′-OH rule of replication turned into a tool.

Since the mid-2000s, next-generation sequencing has read millions of short fragments in parallel and pieced them together with software. That has made a whole human genome affordable to sequence. The same technology, applied to a cell's RNA, is RNA-seq: it measures how much each gene is being expressed. Another way to measure expression is quantitative PCR (qPCR), run on DNA copies of the RNA, which tracks how quickly the product builds up; the more starting copies, the sooner it's detectable.

Editing DNA: CRISPR–Cas9

CRISPR–Cas9 is borrowed from a bacterial defence system against viruses. Cas9 is a DNA-cutting enzyme guided by a short guide RNA. You design the guide RNA to match a 20-base target in the genome. Cas9 cuts only where the guide matches and the target is next to a short sequence called a PAM (NGG for the most-used Cas9).

• Cas9 makes a double-strand break at the target.
• If the cell repairs the break by simply gluing the ends back (non-homologous end joining), it often adds or loses a few bases. That can cause a frameshift and knock out the gene.
• If a repair template with the desired sequence is supplied, the cell can copy it into the break (homology-directed repair), making a precise edit.

Jennifer Doudna and Emmanuelle Charpentier described this RNA-programmable cutting in 2012 and shared the 2020 Nobel Prize in Chemistry. In late 2023 the first CRISPR-based therapy was approved: in the UK for sickle cell disease and β-thalassaemia, and in the USA for sickle cell disease (with β-thalassaemia following in early 2024).

Key terms

PCR

Repeated cycles of heating and cooling that exponentially copy the DNA region between two primers.

Primer

A short single-stranded DNA piece that pairs with the template and gives polymerase a 3′ end to extend.

Taq polymerase

A heat-stable DNA polymerase from Thermus aquaticus, used in PCR.

Gel electrophoresis

Separating DNA fragments by size as an electric field pulls them through a gel toward the positive electrode.

Restriction enzyme / sticky ends

An enzyme that cuts DNA at a specific sequence / the matching single-stranded overhangs it can leave.

Plasmid vector

A small circular DNA molecule used to carry a gene into bacteria.

Transformation

Uptake of outside DNA, such as a plasmid, by a cell.

Blue–white screening

Using an insert's disruption of lacZα, seen with X-gal, to tell recombinant (white) from empty (blue) colonies.

CRISPR–Cas9

A gene-editing system in which a guide RNA directs Cas9 to cut a chosen DNA sequence.

Check yourself

Try answering in your head before you open each answer.

• 1.You start a PCR with 100 copies of a target sequence and run 20 cycles at perfect efficiency. About how many copies do you have? Why would a real reaction give fewer?Show answerHide

  100 × 2²⁰ = 100 × about 1 million ≈ 10⁸ copies. In practice each cycle is slightly less than a full doubling, and in later cycles primers and nucleotides run low, so the reaction slows and levels off.

• 2.You want to put a gene into a plasmid that has an ampicillin-resistance gene and a lacZα gene with an EcoRI site inside it. Your gene has EcoRI sites just outside each end. Outline the steps and how you would find the right colonies.Show answerHide

  Cut both the plasmid and the DNA with EcoRI, mix and add DNA ligase, then transform bacteria. Plate on ampicillin with X-gal (and IPTG). Only plasmid-containing cells survive the ampicillin. Blue colonies have empty plasmids with intact lacZα; white colonies have the gene inserted into lacZα, so pick white ones and check the insert, for example by PCR or sequencing.

• 3.In a PCR, the annealing temperature is set far too low. What would you expect to see on the gel, and why?Show answerHide

  Extra bands of unexpected sizes, possibly a smear. At a low temperature, primers can stick to places where they only partly match, so the polymerase copies unintended regions too. A higher annealing temperature lets primers bind only where they match well.