DNA Sequencing and Genome Databases

Genomes and sequencing

Every cell in your body carries a complete set of instructions. Scientists call the full set of DNA in an organism its genome.

The order of bases is the code. Change one base and a codon may change, then an amino acid, then a protein. That is why knowing the sequence matters in medicine, forensics and evolution.

The Human Genome Project (1990–2003) was an international effort that sequenced the whole human genome. It took over a decade. Today, machines can do the same job in about a day.

How sequencing works: chain termination

The method described here is called chain-termination sequencing. It copies a piece of DNA, but stops the copying at random points. The idea is to make a "ladder" of fragments, each one nucleotide longer than the one before.

You need five things in the reaction tube:

• Template DNA: the single strand you want to read.

• Primer: a short piece of DNA that is complementary to the start of the template. DNA polymerase can only add nucleotides to an existing 3′ end, so the primer gives it a place to start.

• DNA polymerase: the enzyme that builds the new strand by adding free nucleotides, following the base-pairing rules (A–T, C–G).

• Free nucleotides (normal A, T, C, G): the building blocks.

• A small amount of modified nucleotides: these are labelled (for example with a fluorescent dye, a different colour for each base) and they stop chain elongation. Once one is added, no further nucleotide can be joined on.

Because only a small amount of modified nucleotides is present, they are added at random. Sometimes the polymerase adds a normal nucleotide and keeps going. Sometimes it adds a modified one, and that strand stops. Over millions of copies, a strand stops at every possible position.

The new strand is always built in the 5′ → 3′ direction. The template it reads is antiparallel, so it is read 3′ → 5′. You can see how a template and its complementary strand pair up here:

Reading the gel

The mixture of fragments is separated by gel electrophoresis. DNA is negatively charged because of its phosphate groups. When an electric current is applied, the fragments move through the gel towards the positive electrode. Small fragments slip through the gel more easily, so they travel furthest. Large fragments travel only a short distance.

After the run, the fragments are arranged in order of size, differing by exactly one nucleotide. Each ends in a labelled modified nucleotide, and the label tells you which base it is.

To read the sequence, start with the shortest fragment (furthest from the well) and move up the gel. Why the shortest first? The shortest fragment stopped after the very first nucleotide was added to the primer. So its terminating base is the first base of the new strand, at the 5′ end.

A gel that is run for too short a time does not separate the fragments properly. Bands that differ by only one nucleotide stay close together or overlap, so you may miss a base or read it wrongly. This makes the sequence less reliable. Repeating the run, or running it long enough, improves reliability.

Genome databases

When a genome or a gene is sequenced, the sequence is usually uploaded to a genome database. These are publicly available online collections of DNA sequences, which researchers anywhere in the world can search and download for free.

What can scientists do with them?

• Compare sequences. A patient's sequence can be compared with a reference sequence to find a mutation.

• Search for alleles. Researchers can look up the different alleles of a gene and see which ones are linked with a disease.

• Study evolution. Comparing a gene across species shows how closely they are related. The fewer the differences, the more recently they shared a common ancestor.

• Identify genes and design tests. Sequence data helps to find the genes in a new genome and to design primers for PCR or DNA profiling.

Without a database the researcher would not know what the "normal" sequence is. The database also lets them check whether this change has been seen before in other patients.

Interpreting sequences: limits and ethics

A sequence is a starting point, not the full answer.

Sequence is not the same as function

Having the full sequence of a genome does not tell us what every gene does. Much of the genome is non-coding DNA, and it is not obvious from the sequence alone where genes begin and end. A gene's function has to be tested, for example by comparing it with similar genes of known function or by studying what happens when it is changed. Gene activity is also affected by the environment and by epigenetic changes, which a DNA sequence does not show.

Comparing species

Scientists compare the sequence of a gene found in many species, such as the gene for cytochrome c, a protein used in respiration. If the sequence is identical or almost identical in two species, they are closely related. If there are many differences, they are more distantly related. A protein-coding gene is a good marker because every species has it, it does the same job, and changes build up gradually over time.

The amino acid sequences of a protein may be more similar between species than the DNA sequences. This is because the genetic code is degenerate: most amino acids are coded for by more than one codon, often differing only in the third base. A substitution in the third base may leave the amino acid unchanged. These changes show up in the DNA but not in the protein.

Ethical and privacy issues

Your genome contains highly personal information, and also information about your relatives. Concerns include:

• who has access to the data, and how securely it is stored;

• possible discrimination, for example by employers or insurers;

• finding out about a risk of disease that cannot be treated, or that the person did not want to know about;

• whether a person can truly give informed consent for how their data may be used in future.

Back to the start: in the heel-prick example, the lab sequences the gene and compares it with the reference in a genome database. Any difference, such as a substitution, is then checked to see whether it changes the protein and has been linked with the disorder before.

Summary

• The genome is all of an organism's DNA. DNA sequencing works out the order of its bases.

• Chain termination uses template DNA, a primer, DNA polymerase, free nucleotides and a small amount of labelled modified nucleotides that stop chain elongation. This makes fragments differing in length by one nucleotide.

• Fragments are separated by gel electrophoresis. The smallest travel furthest. Read from the shortest to the longest to get the new strand 5′ → 3′. The template is the complement, 3′ → 5′.

• Public genome databases let scientists compare sequences, search for alleles, find mutations and study evolutionary relationships.

• A sequence does not tell us the function of every gene. Amino acid sequences are often more similar than DNA sequences because the genetic code is degenerate.

• Genome data raises ethical issues: privacy, consent, discrimination and unwanted findings.