DNA Manipulation & Profiling

Scientists can read and copy DNA from very small samples - even a single cell, which is especially useful in prenatal diagnosis of genetic disease and in identifying individuals.

In summary: extract the DNA → amplify it (PCR) → separate the fragments (gel electrophoresis) → sequence or profile the result.

EXTRACTING DNA

1. The cells are broken open (lysed) with a salt-and-detergent solution, which emulsifies and breaks down the lipids and proteins of the membranes.
2. The proteins are removed with an enzyme and centrifugation.
3. The DNA is precipitated with ice-cold ethanol and the strands are spooled onto a rod.
4. Impurities are washed away and the DNA is re-dissolved in purified water.

POLYMERASE CHAIN REACTION

A tissue or blood sample is often too small to analyse reliably. The polymerase chain reaction (PCR) solves this by imitating semi-conservative replication in a tube, producing millions of copies of a chosen DNA region. The reaction mixture contains:

1. Template DNA - the sample containing the target segment.
2. Taq polymerase - a DNA polymerase from hot-spring bacteria; because it is heat-resistant, it survives the temperatures used to separate the strands.
3. Free nucleotides - the raw material for the new strands.
4. Primers - short single-stranded nucleic acid sequences (about 6-15 bases) that bind each template strand and determine where copying starts - they 'prime' the DNA to replicate. Primers are often confused with probes, which are used to locate genes.
5. A buffer mix to hold the correct pH, all in a tube inside a programmable thermocycler.

Each cycle then moves through three temperatures:

1. Denature (90-95 °C) - heat separates the double helix into two single-stranded templates.
2. Anneal (55-65 °C) - the sample cools, allowing the primers to bind their complementary sequences.
3. Extend (72 °C) - the optimum for Taq polymerase, which extends each primer, building the new strands. The amount of DNA is doubled.

One PCR cycle. Repeating the cycle doubles the amount of DNA each time - exponential amplification.

When a PCR fails, the temperatures and primers are the usual causes:

GEL ELECTROPHORESIS

Fragments of DNA - made by PCR, or by cutting genomic DNA with restriction enzymes - are loaded into wells in an agarose gel, a matrix riddled with tiny pores, and a voltage is applied across it. DNA (like RNA and proteins, which can be separated the same way) carries a negative charge, so the fragments move toward the positive electrode. Smaller fragments slip through the pores more easily and travel further, so the sample is sorted by size into bands.

A lane of fragments of known sizes - a DNA ladder - is run alongside so that the size of each band can be read off. A labelled probe can identify the band containing a target sequence, and a band can even be cut from the gel, purified and re-amplified.

A gel with a ladder of known sizes and three samples. Samples A and C share the same band pattern - a match.

DNA SEQUENCING

Electrophoresis can be extended to read the actual base sequence, using an automated version of the Sanger method. Special dye-labelled nucleotides stop replication when they are incorporated, producing a set of fragments that differ in length by a single base. Capillary electrophoresis separates the fragments with single-base precision, a laser reads the dye ending each fragment, and software assembles the result into an electropherogram - a trace in which each nucleotide has its own colour peak.

An electropherogram. Each base is flagged by its own dye, so the sequence is read directly from the peaks.

Sequencing data is used to:

1. Read the base sequence of unknown DNA.
2. Detect genetic disease, by comparing a patient's electropherogram with a healthy one.
3. Measure genetic differences within and between species.
4. Establish evolutionary relationships.
5. Map entire genomes - as the Human Genome Project did.

DNA PROFILING

Profiling relies on the fact that individuals differ in their DNA. Members of a species are overwhelmingly similar, however, so scientists target the regions that do vary - and always compare multiple loci before identifying anyone confidently. DNA profiles have special importance in forensic science, such as paternity and criminal cases: a suspect's band pattern can be compared with the pattern from a crime-scene sample.

• Traditional DNA fingerprinting - whole genomic DNA is cut with restriction enzymes (every person's DNA cuts differently), and the fragments are flagged with radioactive or fluorescent probes, giving each person a unique banding pattern on a gel.
• Modern STR profiling - targets short tandem repeats: repeat units of 2-8 bases, repeated over stretches of about 50-300 bases, generally located in introns, at loci spread across the autosomes and the X and Y chromosomes. Everyone has two alleles at each marker - one from each parent - differing in repeat number. The markers are amplified by PCR and separated by capillary electrophoresis; the position of each peak gives the number of repeats.
• VNTR profiling - longer repeats (6-100 bases) exist too, but are harder to interpret and are now little used.

Comparing profiles is used in forensic science, paternity testing and identifying remains. It also raises genuine ethical, economic and cultural questions about who collects and stores genetic data - and who may use it.