Genotyping and Sequencing
Genotyping and sequencing are two of the most commonly performed molecular techniques in the CISAB lab. Both involve DNA extraction and PCR as part of their protocol, and both utilize the technology of a DNA analyzer (The ABI 3730), which is available for use at the Indiana Molecular Biology Institute at IU.
Genotyping is a broad term that can include analysis of microsatellites, restriction fragment length polymorphisms (RFLP), amplified fragment length polymorphisms (AFLP) and more. Most of the work done in the CISAB lab is with microsatellites, which are versatile genetic markers. Microsatellite analysis is based on the concept that different individuals within a population can possess short tandem repeats (1-6 nucleotides) that vary in size at a specific locus within their genome. These variations within the locus can be compared to assess evolutionary relationships and relatedness within a population or between populations.
To perform a microsatellite analysis, PCR is conducted using a fluorescently-labeled primer specific to the “flanking region” surrounding the microsatellite locus of interest. Primers are often “multiplexed” so that several different loci (which can be fluorescently-labeled with different colors for easy identification) are analyzed in a single reaction, saving time and money. Each sample is then mixed with a DNA ladder, which contains DNA fragments of known sizes that are useful for sizing the amplified fragment(s) of interest. The mixture is then run on a DNA analyzer, which characterizes the size of the fragment(s) for each individual using the DNA ladder as a size reference. Software is then used to compare fragments among individuals.
More information about the theory and practice of microsatellites can be found here: Selkoe, K. A., and R. J. Toonen (2006). Microsatellites for ecologists: a practical guide to using and evaluating microsatellite markers. Ecology Letters 9: 615-629.
Sequencing involves determining the exact basepair sequence of a DNA fragment of interest. Major advances in DNA sequencing have come about in the past several years, allowing for a fully automated system of basepair determination that is a lot less labor intensive than previous methods. After PCR of your sequence of interest, the sample is purified to remove any potential contaminants, such as organic residues and high salts. The purified sample is “cycle-sequenced” using a process known as dye-terminator sequencing, in which each base is tagged with a fluorescent dye (e.g., green for A, blue for C, red for T, and yellow for G). The resulting mixture is then purified again and run on the DNA analyzer, which uses a laser to recognize the dyes and outputs a chromatogram, or picture of peaks indicating the sequence and intensity of each dye detected. Software is then used to visualize and compare sequences.