Cot Analysis: Single-copy versus Repetitive DNA

Geoffrey J Graham, University of California at Irvine, Irvine, California, USA

DNA reassociation, analysed by Cot methods, has provided insights into the organization, uneven replication, evolution and expression of genomes, and into disease processes such as viral infection and cancer.

Secondary article
Article Contents
. Introduction . The Measurement and Manipulation of DNA Reassociation . DNA Reassociation Kinetics: Viruses and Bacteria . The Concept of Sequence Complexity . Eukaryotic Genomes: Multiple Kinetic Classes . Stringency of Hybrid Formation: Divergence among Repeated DNAs . Survey of Genomes: Representation of Different Kinetic Classes . Applications of Cot Procedures and Analysis . Rot Curves; Analysis of RNA Populations

Introduction
Native DNA is a double helix: it consists of two antiparallel strands joined noncovalently. (This double-helical DNA is termed duplex DNA.) Under normal physiological conditions, association of the two strands is greatly favoured over dissociation; however, at high temperatures the two strands will spontaneously dissociate. If the temperature is then returned to normal, the single strands will reassociate to form new duplexes. (Reassociation describes the behaviour of the DNA population as a whole. In most cases, the new duplexes are formed by strands that were not originally paired.) Analysis of reassociation rates, and of the thermal stability of resulting duplexes, has yielded valuable information about the types of DNA sequences that are present in genomes and how they are organized. Such analysis is termed Cot analysis. Dissociation of DNA duplexes into single strands is also termed melting or denaturation of the DNA. Association of single strands to form a duplex is also termed hybridization or reannealing of the DNA.

much more rapidly than does one whose reassociation is 50% complete at Cot 5 6000. The DNA of the rst solution can be taken to have much less sequence complexity. By convention, the DNA concentration is expressed in moles of nucleotides per litre and the time is expressed in seconds.

Techniques used in Cot analysis

Performing a DNA reassociation reaction is a simple matter of preparing sheared DNA at a known concentration in a standard aqueous solvent, sealing it into a glass ampule, raising the temperature briey to 1108C to dissociate all duplexes, and incubating the sample in a constant-temperature water bath for a specied time. However, characterization of the resulting duplexes requires several specialized techniques. The information sought

The Measurement and Manipulation of DNA Reassociation

The concept of Cot
The extent to which a DNA sample reassociates during incubation provides information about the DNA sequences it contains. However, this information is much more meaningful if an additional quantity is known: the Cot. At any combination of temperature and solvent that permits DNA reassociation, the extent of reassociation depends on three things: the DNA concentration (Co), the time allowed for reassociation (t) and the sequence organization of the DNA. Since the extent of reassociation is directly proportional to both Co and t, their arithmetic product (Cot) provides a metric against which the extent of reassociation can be compared. A DNA solution whose reassociation is 50% complete at Cot 5 100 reassociates

Usually, two characteristics of the reacted DNA solution are of interest: the amount of reassociation that has occurred, and the thermal stability of the resulting duplexes. The amount of reassociation is of interest because repeated sequences reveal themselves by reassociating more rapidly than single-copy DNA. The thermal stability (stability as the temperature increases) is of interest because duplexes can form between DNA strands that are not a perfect match. However, the greater the mismatch, the lower the temperature needed to reseparate the strands. This depression of dissociation temperature, relative to the dissociation temperature of native DNA, can be used to calculate the percentage of mismatch within populations of related DNA sequences. S1 nuclease Since highly repeated sequences are often interspersed with low-copy or single-copy sequences, reassociated DNA is likely to consist of duplex regions attached to singlestranded tails. These single-stranded tails increase the
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Cot Analysis: Single-copy versus Repetitive DNA

amount of DNA that appears to have formed duplexes. The tails can be removed with S1 nuclease of Aspergillus oryzae, which selectively degrades single-stranded DNA. If the average fragment length is known, comparison of the amount of DNA in duplex structures before and after removal of the tails gives an indication of how repeated and single-copy sequences are interspersed. Hydroxyapatite chromatography Under certain conditions, hydroxyapatite (calcium phosphate hydroxide) binds double-stranded but not singlestranded DNA. Because of this, hydroxyapatite column chromatography can be used to measure both the amount of duplex DNA that has formed in a reassociation reaction, and its thermal stability. The thermal stability of the duplexes can be measured if the column is enclosed in a temperature-controlled water jacket. As the temperature of the column increases, duplex DNA bound to the hydroxyapatite dissociates into single strands, and is then eluted. UV absorption spectroscopy DNA absorbs ultraviolet light (UV light) whose wavelength is near 260 nm. The absorption by DNA increases as the DNA dissociates. The increase in absorption (termed hyperchromic shift) of native DNA varies with its G 1 C composition, but is about 27% for the DNA of higher organisms. As single strands reassociate, their UV absorption decreases (termed hypochromic shift). Changes in UV absorption allow monitoring of DNA dissociation and reassociation in solution. This allows the thermal stability of reassociated DNA duplexes to be measured without hydroxyapatite. Other methods Single-stranded and duplex DNA can be distinguished by both electron microscopy and density-gradient ultracentrifugation (single-stranded DNA is denser). Both techniques have been used to characterize partly reassociated genomes. In addition, radioactively labelled DNA is often used in reassociation experiments. Two specic uses of labelled DNA are described below.

involve radioactively labelling molecules of one of the two populations. The rst method involves physically immobilizing DNA single strands of one of the two populations on a solid support such as nitrocellulose paper. Molecules of the other population are radioactively labelled and remain free to diuse. When the reaction is nished, the nitrocellulose paper is removed from the solution, washed and then analysed for such characteristics as the total amount of bound radioactivity and the thermal stability of the binding (which presumably involves duplex formation between the immobilized DNA on the nitrocellulose paper and radioactively labelled DNA from the solution). The second method is to incubate a small amount of labelled DNA from one population (termed probe) with a large excess of DNA (usually at least a 10 000-fold excess) from the other population (termed driver). The results of the reaction are then monitored by assaying radioactivity. Duplexes formed entirely of probe DNA are negligible in number because the initial concentration of probe is very low and (usually) because most of the probe DNA will form duplexes with driver DNA before it has a chance to form probeprobe duplexes. Duplexes formed entirely of driver DNA are very common in such a reaction, but are not radioactive and hence do not interfere with monitoring of the probedriver duplexes. Shearing of the DNA For the results of Cot reactions to be interpretable, the DNA molecules present at the start of the reaction should be sheared to lengths of 200300 base pairs. If the DNA is not sheared, it will reassociate to form networks whose composition cannot be ascertained. This network formation results from the presence of highly repeated sequences dispersed throughout the genome. Shearing is generally done with the DNA still in duplex form. The best methods for shearing DNA include agitation in a Virtis homogenizer and squirting of the DNA solution through a needle valve at high pressure. Other methods include sonication, treatment with deoxyribonuclease and acid depurination followed by alkaline hydrolysis. The effect of salt concentration The presence of salt in the reassociation solution aects the rate of reassociation. Generally, the higher the concentration of monovalent cations, the faster the reassociation. (Divalent cations such as Mg2 1 are extremely potent accelerators of reassociation, but are usually omitted from reassociation reactions.) The standard solution for measuring reassociation reactions is 0.12 mol L 2 1 sodium phosphate buer, pH 7.0. Reassociation reactions measured at other salt concentrations are expressed in equivalent Cot (Ecot) units.

Practical considerations
Control over hybridization specificity Cot hybridization experiments often involve coincubation of two distinct DNA populations. In these cases, it is usually duplex formation between DNA single strands of dierent populations that is of interest; duplex formation by DNA strands of the same population is an unwanted and confounding side eect. Hence, two methods are used to monitor only the reassociations occurring between single strands of dierent populations. Both methods
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Cot Analysis: Single-copy versus Repetitive DNA

Suppression of DNA composition effects A major potential complication in measurements of DNA thermal stability is its dependence on base composition. Other circumstances being equal, GC-rich DNA dissociates at a higher temperature than does AT-rich DNA. The distribution of GC base pairs throughout genomes is uneven and largely unknown; hence, variations in GC content could mimic or obscure more interesting eects on thermal stability due to base mismatch. The inuence of base composition can be minimized by dissociating the DNA in the presence of 2.4 mol L 2 1 tetraethylammonium chloride (TEACl). Unfortunately, TEACl cannot be used with hydroxyapatite. Other considerations Temperature, pH and DNA fragment length all aect DNA reassociation rates. Their eects must be considered in the planning of reassociation experiments and they must be carefully controlled as the experiment is performed.
Fraction reassociated (%)

0 10 20 30 40 50 60 70 80 90 100 3 10 102 101 1 10 L1 102 s) 103 104 105

Cot (mol

DNA Reassociation Kinetics: Viruses and Bacteria

The genomes of bacteria (and other prokaryotes) and viruses are smaller and simpler than those of eukaryotes. Although prokaryotic and viral genomes do in some cases contain repeats, they consist mostly of sequences that are present only once. Reassociation of such genomes follows simple secondorder kinetics, and the duplexes formed contain little or no base mismatch. Dissociation of reassociated duplexes occurs relatively abruptly as the temperature is raised, and the dissociation prole (percentage dissociation, plotted against temperature) of such duplexes is nearly identical to the dissociation prole of native DNA. Prokaryotic and viral genomes are good experimental materials to investigate DNA reassociation that is not complicated by the presence of multiple kinetic classes. Figure 1 shows a DNA reassociation curve for the genome of the bacterium Escherichia coli.

Figure 1 The time course of reassociation is shown for bacterial and for calf DNA. The amount of reassociation is plotted against the Cot. The time course for bacterial (Escherichia coli) DNA, almost all of which is single-copy DNA, plots as an S-shaped curve (red). If calf DNA were also single-copy DNA, it would plot as a similar curve diplaced to the right (green). The actual curve for calf DNA (blue) has a different shape, however. The shape of the curve indicates that the reaction takes place in two different stages, one early and one late; the midpoints of the two stages (broken vertical lines) are separated by a factor of 100 000. The early stage represents the reassociation of repeated DNA and the later stage that of single-copy DNA. (From Britten and Kohne, 1970).

would suggest, has given rise to the concept of sequence complexity. The sequence complexity of a genome is the total length of the dierent sequences it contains, measured in nucleotide pairs. For species with no repetitive DNA, the sequence complexity is equal to the genome size. The measured complexity of a genome can depend on the criterion at which measurements are performed, because repeats are often imperfect.

Eukaryotic Genomes: Multiple Kinetic Classes

The genomes of higher eukaryotes, both plants and animals, are rich in DNA sequences that reassociate faster than does single-copy DNA. The fastest-reassociating class consists of foldback (also called snapback) sequences. Foldback sequences are sequences within single DNA strands that can form stable duplexes when the single strand folds back upon itself. This reassociation is very rapid because the involved bases are already in close proximity; they need not diuse to each other. A second rapidly reassociating class consists of tandemly repeated sequences that for historical reasons are termed satellite DNA sequences. Individual satellite DNA repeats range in length from a few base pairs to a few thousand. Specic families of satellite DNA repeats can be present in enormous numbers, up to one million or
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The Concept of Sequence Complexity

The fact that one genome contains more DNA than a second genome does not guarantee that the rst genome contains more sequence information. For example, exactly duplicating the 46 chromosomes in a human genome to create a tetraploid genome with 92 chromosomes would not increase its sequence information. The fact that some genomes contain large numbers of repeated sequences, and thus contain less sequence information than their size

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Cot Analysis: Single-copy versus Repetitive DNA

more copies per haploid genome. On the other hand, many families of tandem repeats are much smaller, containing as few as two members. Interspersed repeats form a third type of rapidly reassociating sequence. They are usually derived from transposable elements, although some simple-sequence interspersed repeats may arise spontaneously. Interspersed repeats range in number from only a few copies per haploid genome to hundreds of thousands of copies or more. The Alu interspersed repeat of the human genome, for example, is present in roughly 500 000 copies. Interspersed repeats are interspersed with low-copy or single-copy sequences and with each other. To a much lesser extent, they are also interspersed with satellite DNA repeats. A fourth category of repeated sequences are low-copy long repeats. The genes encoding the pigments for human redgreen colour vision, for example, are present in long, tandem repeats of 39 kilobase pairs. Each human X chromosome contains between two and six copies of the repeat (Nathans et al., 1992). A fth kinetic class consists of sequences that are present only once per haploid genome. These are single-copy sequences. Figure 1 illustrates how Cot curves revealed the existence of repetitive DNA. Calf DNA reassociates much more rapidly than it would if calf DNA were entirely single copy, and the reassociation curve has a shape indicative of at least two components.

been repeated cycles of expression and sequence drift within specic families. Divergence among satellite or interspersed repeats can be measured by Cot analysis. Diverged sequence families will reassociate to form duplexes containing mismatched base pairs. The greater the average divergence within a family, the more mismatches will occur in the reassociated duplexes. This mismatch can be estimated from the reduction in dissociation temperature that the reassociated duplexes show, compared with native DNA. In a typical measurement, a radioactively labelled recombinant DNA clone containing a member of the repeat family is dissociated, mixed with a great excess of dissociated genomic DNA, and allowed to reassociate. The dissociation prole of the reassociated duplexes is then compared with the dissociation prole of the native recombinant DNA clone. Very large DNA repeat families tend to contain many diverged members, suggesting perhaps that signicant time is needed for sequence families to acquire very many copies. Nevertheless, families of the same size can have very dierent average divergences. Less-divergent families are usually thought to be newer in origin; however, it is possible instead that divergence has been suppressed by natural selection or some other inuence.

Survey of Genomes: Representation of Different Kinetic Classes

Eukaryotic species dier in the distribution of their DNA into various kinetic classes. Some lower eukaryotes, such as the fungi Neurospora crassa and Aspergillus nidulans, contain very little repetitive DNA aside from a few repeated gene families such as the ribosomal RNA gene families. In other simple eukaryotes, such as dinoagellates and trichomonads, there are abundant repeats. In some higher eukaryotes, the amount of satellite DNA is less than a few per cent of the genome, while in others (such as Drosophila nasutoides) it is about 60%. The amount of interspersed repetitive DNA also varies between species. The interspersion pattern of a genome is the overall pattern in which repeated and single-copy (or low-copy) sequences are mutually interspersed. Most species, including humans, have a short period interspersion pattern. In the toad Xenopus laevis, for example, the repeated sequences average 300 base pairs in length and are interspersed with single-copy sequences of about 900 base pairs in length, throughout most of the genome. Other species have a long period interspersion pattern. In the fruit y Drosophila melanogaster, for example, the repeated sequences average 50006000 base pairs in length and are interspersed with single-copy sequences at least 13 000 base pairs in length.

Stringency of Hybrid Formation: Divergence among Repeated DNAs

In nearly all cases, the members of a given DNA repeat family are descended from a common ancestor sequence. Since the mechanisms that create multiple copies of a single DNA sequence are precise, it seems likely that such copies are identical or very similar when created. However, the members of present-day DNA repeat families often dier markedly in sequence. The reason is that genetic changes (base substitutions, insertions and deletions) have occurred independently in the various family members since their creation. The various family members are said to have drifted or diverged in sequence. The events that create tandemly repeated DNA sequence families are probably quite dierent from the events that create interspersed repeat families. Nevertheless, both types of repeat sequence appear to be created by processes that are recurrent. Repeated sequence families of both types are often organized into subfamilies, where there is signicantly more similarity within subfamilies than between subfamilies. These subfamilies give the impression that there have
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Cot Analysis: Single-copy versus Repetitive DNA

Still other species have what could be termed an intermediate period interspersion pattern. Among these are chickens and ducks.

Applications of Cot Procedures and Analysis

Purification of DNA sequence classes
Cot hybridization is often used as a preparative technique. DNA is reassociated to a certain Cot value, the duplexes are separated from the single strands, and one of the two fractions is analysed or used in some other way. Cot hybridization has been used to isolate foldback sequences, for the purpose of determining their relative sensitivity in vivo to the mutagen nitrosoguanidine. It has been used to isolate various populations of repeated sequences, for the purpose of learning the frequencies with which their cytosine residues are methylated in vivo. It has been used to isolate satellite DNA repeats for use in chromatin reconstitution experiments. It has often been used to select certain types of sequences to be cloned and studied; the human Alu element and the mouse minor satellite (probably the functional sequence in mouse centromeres) were discovered and isolated this way. Cot hybridization is also used to isolate fractions of DNA for use in uorescence in situ hybridization (FISH) experiments. In some experiments, DNA repeats isolated by Cot fractionation are used to paint chromosomes. In other cases, the repeats are used as blocking agents to suppress background signals in FISH hybridizations (Landegent et al., 1987). In still other cases, repeats isolated by Cot fractionation and attached to biotin moieties (which allows their easy removal from solution) are used to remove the repeats from a probe population (Craig et al., 1997).

cated. On the other hand, the chorion gene region of D. melanogaster is overreplicated at least 10-fold in late-stage egg chambers. There are many species where ribosomal genes are overreplicated in some tissue or developmental stage. In addition, human cancers often contain overreplicated regions of DNA that aect their behaviour. Measurement of uneven replication is done by DNA hybridization and Cot analysis.

Measurement of DNA sequence polymorphism

The extent of DNA sequence polymorphism within a species has important implications for how species evolve. Cot analysis is routinely used to measure this. An experiment to measure intraspecic DNA sequence polymorphism is illustrated in Figure 2. A very important application of Cot analysis has been the investigation of interspecies relationships. Over time, the genomes of related species diverge in sequence. Dissociation proles of hybrid duplexes from the two species can determine their average mismatch and thus their average divergence. From this, the time since the species diverged can be estimated. When multiple related species are examined, phylogenetic trees can be constructed (Sibley et al., 1990).

0 Fraction of DNA in duplex form 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 40 45 50 55 60 65

Quantification of specific DNA sequences

Cot analysis is useful for measuring the concentration of any specic DNA sequence within a population. It has been used to learn the number of genomically integrated virus copies in infected cells (Green et al., 1976), the number of integrated plasmid copies in transfected cells, the number of genes in multigene families, and the amount of human DNA in rodenthuman cell hybrids. It has been used to determine the ploidy (the number of complete chromosome sets) in species where the chromosomes are not clearly distinguishable. It has also been used to conrm suspicions that bacteriophage Mu incorporates a piece of host DNA at one end (Daniell et al., 1975.) The replication of genomes sometimes occurs unevenly. In the giant salivary gland polytene chromosomes of D. melanogaster, satellite DNA sequences are underrepli-

Temperature (C)
Figure 2 Dissociation profiles reveal sequence divergence within a species. For simplicity, the species is considered to be haploid and to have only single-copy DNA sequences. In assay no. 1, DNA from several individuals is dissociated, reassociated separately to a specified Cot value, mixed and then subjected to a dissociation profile (red line). In assay no. 2, the DNA samples are first mixed, then dissociated, reassociated to the specified Cot value and subjected to a dissociation profile (blue line). In assay no. 1, all duplexes are composed of single strands from the same individual. In assay no. 2, most duplexes (the fraction depending on the number of individuals tested) are composed of single strands from different individuals. The duplexes derived from different individuals contain base mismatches absent from duplexes derived from a single individual. The decrease in thermal stability that occurs when the DNA is from different individuals gauges DNA sequence polymorphism within the species. This technique also has been used to gauge sequence divergence in diploid species containing repetitive DNA.

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Cot Analysis: Single-copy versus Repetitive DNA

Often, interspecies comparisons are restricted to singlecopy and low-copy DNA. This genomic fraction contains most of the genes and its Cot behaviour is relatively easy to interpret. However, highly repetitive sequences have also been used in interspecies comparisons. Cot analysis of closely related species has also shed light on evolution itself. The sequence divergence between humans and chimpanzees, for example, is less than 2% overall and only about 0.5% in the active coding sequences of functional nuclear genes. These surprisingly low values limit explanations of what the genetic dierence between humans and chimpanzees is, and how it evolved. In recent years, Cot analysis has been used to address more subtle evolutionary questions. In one study of parasitic tapeworms in ocean sh, in which seven tapeworm species are each highly specic to a single host, it was concluded that there is much less genetic variation between the tapeworm species than between the hosts (Verneau et al., 1997). This suggests that the tapeworm species have not coexisted with their hosts for long evolutionary periods, and that there has instead been host-switching in relatively recent times. Although direct analysis of DNA sequences has largely replaced Cot analysis in evolutionary studies, in some cases the two methods complement each other. In a recent construction of a phylogenetic tree for stork species (Slikas, 1997), DNADNA hybridization measurements and comparisons of a mitochondrial gene sequence each provided information that the other method could not. DNADNA hybridization measurements resolved relationships between distantly related species, but could not resolve relationships between more closely related species. Sequence comparisons of the mitochondrial gene used (cytochrome b) resolved relationships between closely related species, but not between more distantly related species.

A fourth is to measure the concentration of specic sequences of interest, such as the amount of a tumour virus RNA present in tumours (Jaenisch et al., 1975). Cot technology has largely been replaced by DNA sequencing. Although DNA sequence information is harder to obtain, it allows weak relationships between sequences to be detected more reliably. It also allows results from dierent experiments and laboratories to be compared, and it provides more clues about DNA function. Nevertheless, as the examples above indicate, there are still important uses for Cot techniques.

References
Britten RJ and Kohne DE (1970) Repeated segments of DNA. Scientic American 222(4): 2431. Craig JM, Kraus J and Cremer T (1997) Removal of repetitive sequences from FISH probes using PCR-assisted anity chromatography. Human Genetics 100(34): 472476. Daniell E, Kohne DE and Abelson J (1975) Characterization of the inhomogeneous DNA in virions of the bacteriophage Mu by DNA reannealing kinetics. Journal of Virology 15(4): 739743. Galau GA, Klein WH, Davis MM et al. (1976) Structural gene sets active in embryos and adult tissues of the sea urchin. Cell 7(4): 487505. Green MR, Chinnadurai G, Mackey JK and Green M (1976) A unique pattern of integrated viral genes in hamster cells transformed by highly oncogenic human adenovirus 12. Cell 7(3): 419428. Jaenisch R, Fan H and Croker B (1975) Infection of preimplantation mouse embryos and of newborn mice with leukemia virus: tissue distribution of viral DNA and RNA and leukemogenesis in the adult animal. Proceedings of the National Academy of Sciences of the USA 72(10): 40084012. Landegent JE, Jansen in de Wal N, Dirks RW, Baas F and van der Ploeg M (1987) Use of whole cosmid cloned genomic sequences for chromosomal localization by non-radioactive in situ hybridization. Human Genetics 77(4): 366370. Nathans J, Merbs SL, Sung C-H, Weitz CJ and Wang Y (1992) Molecular genetics of human visual pigments. Annual Review of Genetics 26: 403424. Sibley CG, Comstock JA and Ahlquist JE (1990) DNA hybridization evidence of hominid phylogeny: a reanalysis of the data. Journal of Molecular Evolution 30(3): 202236. Slikas B (1997) Phylogeny of the avian family Ciconiidae (storks) based on cytochrome b sequences and DNADNA hybridization distances. Molecular Phylogenetics and Evolution 8(3): 275300. Verneau O, Catzeis FM and Renaud F (1997) Molecular relationships between closely-related species of Bothriocephalus (Cestoda: Platyhelminthes). Molecular Phylogenetics and Evolution 7(2): 201207.

Rot Curves; Analysis of RNA Populations

Although RNA is usually a single-stranded nucleic acid, RNA molecules can form duplexes either with each other or with DNA. The ability of RNA to participate in RNA/ DNA duplex formation allows RNA populations to be analysed by Cot methods. Such analysis is termed Rot analysis. One use of Rot analysis is to determine the total fraction of the genome that is transcribed in a given cell type (such as unfertilized oocytes). A second use is to determine the relative frequencies of RNA populations in cells (Galau et al., 1976). A third use is to characterize sequence divergence within populations of similar RNA molecules.

Further Reading
Britten RJ and Davidson EH (1976) Studies on nucleic acid reassociation kinetics: empirical equations describing DNA reassociation. Proceedings of the National Academy of Sciences of the USA 73(2): 415419. Britten RJ, Graham DE and Neufeld BR (1974) Analysis of repeating DNA sequences by reassociation. Methods in Enzymology 29: 363 418.

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