Molecular Methods for Evolutionary Genetics
We are entering a particularly fruitful period in evolutionary genetics, as rapid technological progress transforms the investigation of genetic variation within and between species. Molecular Methods for Evolutionary Genetics is a collection of advanced
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1. Introduction The original motivation for the development of DNA microarrays was the detection of genome sequence variation (1, 2). Although myriad applications of DNA microarrays were subsequently developed, including the analysis of mRNA expression levels (3), protein– DNA interactions (4), and genome amplifications and deletions (5, 6), the discovery and analysis of single-nucleotide polymorphism (SNP) variation using microarrays has remained a mainstay of modern molecular genetics. All DNA microarray methods for detecting sequence differences rely on the chemistry of DNA duplex formation. Under appropriate reaction conditions, duplexes that are perfectly complementary in their DNA sequence are strongly favored over duplexes that contain one or more mismatched bases. In a typical
Virginie Orgogozo and Matthew V. Rockman (eds.), Molecular Methods for Evolutionary Genetics, Methods in Molecular Biology, vol. 772, DOI 10.1007/978-1-61779-228-1_10, © Springer Science+Business Media, LLC 2011
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experiment, the efficiency of duplex formation is measured by labeling a DNA sample with a fluorophore and quantifying the fluorescent signal at thousands to millions of probes following a hybridization reaction. Sample DNA fragments that are perfectly complementary to the probe sequence will exhibit maximal fluorescent signals, whereas the presence of even a single base difference that reduces complementarity results in diminished signals. For the purpose of DNA sequence comparison, it is necessary to maximize the difference in efficiency of matched and mismatched duplex formation. Although several factors determine the efficiency of these reactions, two factors dominate. The first is the position of the mismatched base within the probe. Empirical studies have shown that mutations corresponding to mismatches in the central portion of the probe have the greatest impact on hybridization efficiency (7, 8). The second is the relationship between the predicted probe melting temperature (Tm) and the temperature at which the hybridization reaction is performed (9). The Tm of a probe is defined as the temperature at which 50% of the DNA molecules are in a duplex state. Previously, we performed a systematic study of the relationship between probe Tm and the discriminatory power of a probe sequence. We found that a probe melting temperature 2–5°C lower than the temperature at which hybridization is performed maximizes the sensitivity of duplex formation to single-base mismatches (9). Here, I describe guidelines for designing DNA microarrays that employ these two simple principles. The manufacture of DNA microarrays corresponding to these designs is best realized using commercial manufacturers such as Agilent, Nimbelgen (Roche), or Affymetrix. These designs are well suited to SNP discovery on a genome scale (7) or targeted SNP genotyping of either individuals or pools of millions of individuals (10). I provide methodological details on preparation of samples and DNA hybridization experiments. The detailed steps
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