Molecular Evolution: Techniques


Progress in the field of molecular evolution has been intricately tied to advances in the techniques used to characterize the molecular basis of genetic change. Although DNA sequencing currently provides the most information on evolutionary processes at the molecular level, techniques such as immunological distances, protein sequencing, protein electrophoresis, DNA hybridization, and restriction fragment analyses contributed to the development of more advanced techniques and continue to have important usages.

Keywords: DNA hybridization; sequencing; immunological distances; restriction enzymes; molecular evolution

Figure 1.

The principles of gel electrophoresis. (a) Image from a stained starch gel showing the gene products from two loci of malate dehydrogenase, which is a dimeric enzyme (i.e. it is composed of two subunits), as indicated by the presence of heteropolymer bands in heterozygotes at both of the loci (indicated by the arrows). In lanes 1 and 2, the individuals are heterozygous for two alleles at both Mdh‐1 (bands A and B) and Mdh‐2 (bands A′ and B′). In lanes 3–6, the individuals are heterozygous at Mdh‐1 and homozygous at Mdh‐2 (band A′ only). Isozyme analysis might compare how many loci were expressed in different species, while allozyme analysis would be more concerned with alleles within loci. (b) Agarose gel showing PCR products amplified using primers designed to recognize the gene for superoxide dismutase (SOD). Lane 5 (arrow) shows the presence of multiple‐size products amplified using the same primers. The larger band (800 bp) migrates more slowly through the gel matrix than the smaller band (350 bp).

Figure 2.

The principles of DNA hybridization and primer‐mediated DNA extension. (a) Double‐stranded DNA (dsDNA) is ‘melted’ to single strands by heating to a sufficient temperature to break the hydrogen bonds that hold the two strands of the double helix together. The temperature required to melt the strands in a homoduplex molecule (between completely matching complementary strands) depends on the nucleotide composition. Complementary base pairing between guanine (G) and cytosine (C) nucleotides involves triple hydrogen bonds, whereas base pairing between adenine (A) and thymidine (T) nucleotides involves double bonds. G‐C rich regions therefore require higher temperatures to melt than A‐T rich regions. Base‐pair mismatches (i.e. in heteroduplex molecules) also result in lowering of the melting temperature. (b) Both the base composition and the degree of mismatch between a primer and a particular target region of DNA determine the stringency of the reaction conditions that will allow annealing of the primer to the 5′ region of the complementary strand of template DNA. The more mismatches there are, the lower the temperature at which the primer will anneal and the lower the specificity of the reaction (i.e. the higher the chance that the primer will anneal to nontarget regions of DNA). Extension from the 3′ end of the primer (arrow) occurs via the action of DNA polymerase, which catalyses the incorporation of free dNTPs into the growing chain (indicated in bold type) using the complementary target strand as a template.

Figure 3.

The principles of DNA sequencing. (a) Autoradiograph from a manual sequencing gel (denaturing polyacrylamide gel). Each numbered lane is subdivided into four lanes corresponding to each of the nucleotide types (G, A, T, C). In each lane, the DNA fragments are terminated when a ddNTP rather than a dNTP is incorporated into the synthesized chain. The smallest fragments (closest to the primer) are at the bottom of the gel; the largest fragments are at the top. Sequences are read up the gel (bottom to top) from left to right. The sequence from the boxed region of lane 3 is indicated. Note that lane 4 is the sequence from the PCR product containing multiple size fragments in Figure b; heterogeneous DNA regions result in unreadable DNA sequences. (b) Chromatograph from an automated sequencing gel. Each nucleotide type in this case is labelled with a different fluorescent dye, producing intensity peaks of different colours for fragments passing a stationary laser (also run on a denaturing polyacrylamide gel). The image is directly recorded into a computer rather than being read manually from the gel. The nucleotide sequence is shown at the top of the output, with the base pair position indicated immediately below. The scale on the left indicates the fluorescent intensity. Note that at base pairs 21 and 22 the computer is unable to distinguish which nucleotides are present (indicated by ‘N’) because two peaks are overlapping (for a heterogeneous sequence the entire sequence would appear in this way), but the rest of the sequence is unambiguous.



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Further Reading

Adams MD, Fields C and Venter JC (eds) (1994) Automated DNA Sequencing and Analysis. San Diego: Academic Press.

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Mable, Barbara K(Apr 2001) Molecular Evolution: Techniques. In: eLS. John Wiley & Sons Ltd, Chichester. [doi: 10.1038/npg.els.0001796]