3.2 Applications and skills
3.2.2 Nature of Science: Technique and technologies
A recurring theme in the Nature of Science is that developments in research and scientific understanding follow improvements in technique and technologies.
Autoradiography and Cairn’s technique
- Radioactive isotopes decay slowly, releasing particles that appear as black dots on X-ray film. The resulting pictures are called autoradiographs.
- Autoradiography is a technique used in molecular biology to identify the locations and shapes of structures, or to track the movement of molecules in vivo over time.
- In 1963, John Cairns demonstrated, using autoradiography, that bacterial chromosomes are circular, and that Y-shaped replication forks are present during DNA replication.
- In Cairns’ method, E. coli bacteria are cultured in a medium containing thymidine that has been labelled with a radioactive isotope of hydrogen, tritium (3H).
- During DNA replication, the tritiated thymidine is incorporated into the bacterial chromosome as radioactive thymine.
- Thin sections of labelled cells are covered with X-ray film. As the tritiated thymine decays, beta particles expose the film in patterns indicating the shape of the DNA molecule.
- The Cairns technique (labelling with tritiated thymidine followed by autoradiography) can also be used to get information about eukaryotic chromosomes and to measure the length of DNA molecules.
sequencing and databases
- Gene sequencing involves a combination of analytical techniques and high-speed computer processing in order to determine the exact base sequence (A, T, C and G) of DNA in a sample.
- A typical method of sequencing genes involves the following technologies:
- labelling nitrogenous bases with a fluorescent marker molecule
- using lasers and optical imaging technology to detect types of fluorescence at different locations on the DNA molecule
- processing the information using specialised computer hardware and software.
- Much of the data obtained by large-scale sequencing projects is held in public databases that can be accessed by scientists from anywhere in the world.
- Using databases, a researcher may:
- search for information on a gene locus and the protein products of that gene
- compare amino acid sequences in homologous genes
- search for similar sequences in different species
- compare sequences of genes in more than one species
- deduce evolutionary relationships based on the number of differences in sequences of different genes.
Skill: Using databases
- Required database: Online Mendelian Inheritance in Man (OMIM)
- Open the home page www.omim.org. Click on Gene Map. Search by chromosome number, gene name (locus) or product. Some examples are listed below:
Cyclin protein (cell cycle regulation)
- Required database: National Centre for Biotechnology Information (NCBI) – Genbank: www.ncbi.nlm.nih.gov/genbank/
- This database stores amino acid sequences and nucleotide sequences. However, extra software is required to perform the comparison.
- The image above shows a portion of the amino acid sequence of the NBN protein in 23 different species.
- Each amino acid is represented by a different letter. Amino acids with similar chemical properties are shown in the same colour. For example, A, F, I, L and V have non-polar R groups, and are shown in blue.
- The graph at the bottom shows the level of whether the amino acid residue is the same at that position. An asterisk (*) means there is 100% conservation at that position for the species investigated.
- Nibrin proteins in humans (Homo) and chimpanzees (Pan) have the same number of amino acids, 754.
- Print a copy of Figure 3.2.2c. Determine the number of differences in the amino acid sequence between i) humans and chimpanzees and ii) two other species.
Thymidine is the precursor to thymine.
Figure 3.2.2f – Films exposed
Films used in autoradiography provide permanent images.
Why not collect data from databases for your internal assessment investigations? What kinds of research questions can be answered using genome information?
Calculate the length of the prokaryotic chromosome in Figure 3.2.2a.
- Autoradiography is/was also used to:
- label DNA during PCR and gel electrophoresis 3.1.5
- produce X-ray diffraction images 2.2.6
- provide evidence for the Calvin cycle 8.2.3 (HL)
- study translocation in plants 9.2.4.
- Learn more about how molecular evidence is used to determine evolutionary relationships in 5.1.4.
- HL students will learn more information on gene sequencing in 7.2.1.
- Learn how to perform a multiple sequence alignment in Option B 13.1.5.