DNA Sequencing and Genome Databases
A baby in Cork has a heel-prick test a few days after birth. If the result suggests PKU (phenylketonuria), a lab may sequence one gene and compare it with a reference sequence held in a database. How can anyone "read" DNA, and how does a database turn one patient's sequence into a diagnosis? By the end of this page you will be able to answer both.
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.
Define the terms genome* and DNA sequencing*.
Define the terms genome and DNA sequencing.
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:
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Template DNA: the single strand you want to read.
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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.
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DNA polymerase: the enzyme that builds the new strand by adding free nucleotides, following the base-pairing rules (A–T, C–G).
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Free nucleotides (normal A, T, C, G): the building blocks.
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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:
A sequencing method uses template DNA, primer, DNA polymerase, free nucleotides and a small amount of modified nucleotides that stop chain elongation. a) State the role…
A sequencing method uses template DNA, primer, DNA polymerase, free nucleotides and a small amount of modified nucleotides that stop chain elongation.
- a) State the role of the primer.
- b) Explain why the modified nucleotides produce fragments differing in length by one nucleotide.
- c) Explain why the fragments need to be labelled in order to read the sequence.
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.
In a DNA sequencing gel, fragments are separated by size. a) State the technique used to separate the fragments. b) State which fragments move furthest through the gel.…
In a DNA sequencing gel, fragments are separated by size.
- a) State the technique used to separate the fragments.
- b) State which fragments move furthest through the gel.
- c) State how the fragments differ from each other.
In a sequencing reaction, a newly synthesised strand is built from a template. Fragments of the following lengths were found on a gel, each ending in a labelled…
In a sequencing reaction, a newly synthesised strand is built from a template. Fragments of the following lengths were found on a gel, each ending in a labelled terminating nucleotide:
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1 nucleotide: T
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2 nucleotides: A
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3 nucleotides: G
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4 nucleotides: G
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5 nucleotides: C
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6 nucleotides: A
- a) Write the sequence of the newly synthesised strand in the order $5' \to 3'$.
- b) Write the sequence of the template strand it was made from, in the order $3' \to 5'$.
- c) Explain why the shortest fragment is read first.
The following fragments were read from a gel, from shortest to longest, with the terminating base for each: C, A, T, T, G, C, A, G. a) Deduce the sequence of the new…
The following fragments were read from a gel, from shortest to longest, with the terminating base for each: C, A, T, T, G, C, A, G.
- a) Deduce the sequence of the new strand in the order $5' \to 3'$.
- b) Deduce the complementary template sequence and state its polarity.
- c) Explain what the result would show if the gel were run for too short a time, and how this would affect the reliability of the sequence.
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?
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Compare sequences. A patient's sequence can be compared with a reference sequence to find a mutation.
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Search for alleles. Researchers can look up the different alleles of a gene and see which ones are linked with a disease.
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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.
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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.
Name two uses of publicly available genome databases.
Name two uses of publicly available genome databases.
A researcher at a university in Dublin compares the sequence of a gene from a patient with the reference sequence in a genome database: Reference: 5′-ATGGCCTTAAGC-3′…
A researcher at a university in Dublin compares the sequence of a gene from a patient with the reference sequence in a genome database:
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Reference: 5′-ATGGCCTTAAGC-3′
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Patient: 5′-ATGGCCATAAGC-3′
- a) Identify the type of mutation and its position.
- b) Explain how a database helps the researcher in this comparison.
- c) State one way in which this type of mutation could have no effect on the protein.
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:
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who has access to the data, and how securely it is stored;
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possible discrimination, for example by employers or insurers;
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finding out about a risk of disease that cannot be treated, or that the person did not want to know about;
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whether a person can truly give informed consent for how their data may be used in future.
Scientists compare the gene for cytochrome c in humans, chimpanzees and yeast stored in a genome database. The amino acid sequences of the encoded protein are identical…
Scientists compare the gene for cytochrome c in humans, chimpanzees and yeast stored in a genome database. The amino acid sequences of the encoded protein are identical (0 differences) in human and chimpanzee, although their DNA sequences differ at a few positions; between human and yeast, both the DNA and the amino acid sequences differ at many positions.
- a) Interpret these results in terms of evolutionary relationships.
- b) Explain why a protein-coding gene is a good marker for comparing species.
- c) Explain why the amino acid sequences may be more similar between species than the DNA sequences, using the properties of the genetic code.
Next-generation sequencing machines can sequence an entire human genome in a day, whereas the Human Genome Project took over a decade. a) Explain why a sequenced genome…
Next-generation sequencing machines can sequence an entire human genome in a day, whereas the Human Genome Project took over a decade.
- a) Explain why a sequenced genome alone does not tell us the function of every gene.
- b) Discuss two benefits of the availability of sequence data in public databases to medicine.
- c) Discuss one ethical or privacy concern arising from the sequencing of individuals' genomes.
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
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The genome is all of an organism's DNA. DNA sequencing works out the order of its bases.
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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.
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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′.
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Public genome databases let scientists compare sequences, search for alleles, find mutations and study evolutionary relationships.
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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.
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Genome data raises ethical issues: privacy, consent, discrimination and unwanted findings.