1Gel electrophoresis

DNA samples are placed in wells at one end of a slab of gel, and an electric current is run through it. Because of its phosphate groups, DNA carries a negative charge and moves towards the positive electrode. The gel is a mesh: short fragments slip through it quickly, long fragments are held back. So after a set time, fragments are sorted by length, the shortest furthest from the wells. Each band is many copies of fragments of the same length. A DNA ladder, a mix of fragments of known lengths, is run in one lane so that the length of other bands can be read off by comparison.

In Figure 1, sample 1 has bands level with the 700 and 300 base pair (bp) rungs of the ladder, so it contains fragments of about 700 bp and 300 bp. Sample 3 shares the 700 bp band but has a 400 bp band instead of 300.

The CED says electrophoresis separates fragments "by size and charge". For DNA, every nucleotide carries the same charge, so charge per unit length is about the same for all fragments; it is the charge that makes DNA move at all, and the size that decides how far.

2PCR: copying one stretch of DNA

PCR copies a chosen region of DNA, the region between two primers. Each cycle has three steps:

1. Denaturation. The mixture is heated to about 95 °C. The hydrogen bonds between bases break and the two strands separate.
2. Annealing. It is cooled so that the primers, short single strands made to match the two ends of the target region, pair with the separated strands.
3. Extension. A heat-stable DNA polymerase adds nucleotides to the 3′ end of each primer, building a new complementary strand.

Each cycle doubles the number of copies of the target. Starting from N copies, after n cycles there are

$$\text{copies} = N \times 2^{n}$$

Worked example. A forensic sample contains 4 copies of a target region. How many after 10 cycles? 4 × 2¹⁰ = 4 × 1,024 = 4,096. After 30 cycles, a single starting copy becomes 2³⁰, about 1.07 × 10⁹, over a billion copies: enough to see as a band on a gel. That is why PCR lets a trace of blood or a single hair be analysed.

PCR uses the same rules as replication (topic 6.2): separate the strands, provide a primer, let DNA polymerase build 5′ to 3′.

3Bacterial transformation

Transformation introduces foreign DNA into bacterial cells. Usually the DNA is a plasmid carrying the gene of interest and a gene for resistance to an antibiotic. The bacteria are treated (for example with calcium ions and a brief heat shock) so that some take up the plasmid. Only a small fraction do.

To find those that did, the cells are spread on a plate containing the antibiotic. Only cells that took up the plasmid, and so carry the resistance gene, can grow. Every colony on that plate is transformed. Because the genetic code is shared (topic 6.4), the bacteria can transcribe and translate a gene from a completely different organism, such as a human insulin gene.

A full transformation experiment has controls, and predicting the plates is a standard question.

Transformation efficiency measures how well it worked: the number of transformed colonies per microgram of plasmid DNA spread on the plate.

Worked example (invented data). 0.1 µg of plasmid is mixed with bacteria in a final volume of 500 µL. 100 µL is spread on a plate with antibiotic, and 84 colonies grow.

• DNA spread = 0.1 µg × (100 ÷ 500) = 0.02 µg.
• Efficiency = 84 ÷ 0.02 = 4,200 transformants per µg.

The trap is using the whole 0.1 µg: only one fifth of the mixture was spread.

Transformed bacteria divide and copy the plasmid each time, so a whole colony is a clone of the gene. This is gene cloning: propagating a DNA fragment by letting cells copy it. Organisms that carry a gene from another species are transgenic, a kind of genetically modified organism; the term covers transgenic animals and plants as well as bacteria.

DNA is negative and runs to the positive electrode, short fragments furthest; PCR doubles the target every cycle (N × 2ⁿ); after transformation, only cells that took up the plasmid grow on the antibiotic plate.

4DNA sequencing and DNA fingerprints

DNA sequencing determines the order of nucleotides in a DNA molecule. Modern machines read millions of fragments at once. Sequences from different samples can then be compared base by base.

Comparing DNA, whether as gel bands or as sequences, gives a DNA fingerprint (or profile), and it is used in three ways the CED lists.

• Forensic identification. Regions that vary a lot between people are copied by PCR and separated by electrophoresis. A suspect whose bands all match those from a crime scene could be the source; one whose bands do not match is excluded. A mismatch is much stronger evidence than a match, because a match could, rarely, be chance.
• Identifying organisms. Amplifying and sequencing a short, standard gene can identify a species from a scrap of tissue, for example to check whether fish sold as one species is really another.
• Phylogenetic analysis. The fewer differences between two species' sequences, the more recently they shared an ancestor (Unit 7).

Worked example (invented sequences). A 20-base region is compared:

P and Q differ at 1 position (position 12); Q and R at 3 (positions 5, 9 and 17); P and R at 4. So P and Q are the most closely related, and R branched off earlier.

Paternity. Each band in a child's profile must come from the mother or the biological father. Remove the bands the child shares with the mother; every remaining band must appear in the father's profile.