Linda K. Park1, Paul Moran1, and Deborah A. Nickerson2
1Coastal Zone and Estuarine Studies Division
Northwest Fisheries Science Center
National Marine Fisheries Service
2725 Montlake Boulevard East
Seattle, WA 98112
2Department of Biotechnology
University of Washington FJ 20
Seattle, WA 98195
The most time-consuming and costly part of research in molecular population genetics of Pacific salmon is the identification and characterization of specific polymorphisms that may be useful for stock discrimination. However, after useful markers have been identified, the bulk of the research effort will focus on assaying large numbers of individuals for those markers to establish baseline data on genetic variation. Many of the methods that are traditionally used for the initial research are fairly rapid on a small scale (i.e., assaying fewer than 50 individuals at a time) but are inefficient on a larger scale in terms of both time and money. When it becomes necessary to assay a hundred individuals for multiple loci in each of 50 populations, many standard DNA methods quickly reach a bottleneck. Efficient, large-scale assaying will require a different approach to the detection of polymorphism.
This report summarizes the result of the application of a recently developed method for rapidly assaying specific polymorphisms in large numbers of individuals. This method increases the screening potential of a single laboratory by one or two orders of magnitude. The oligonucleotide ligation assay (OLA) was developed by one of us (D. Nickerson) for use in human genetics research associated with the Human Genome Project (Nickerson et al. 1990). This study represents a collaborative effort to develop this assay for use in salmon genetics. Here, we describe the assay and give an example of how it is applied to the study of chinook salmon populations in the Snake River.
This particular assay relies on a class of enzymes known as ligases, which join, or ligate, two pieces of DNA to form one continuous strand. Ligases are found in most cells as part of the natural repair mechanism for DNA. DNA can be damaged by UV light or by exposure to certain chemicals. Typically, ligases are used by the cell to repair breaks in the sugar-phosphate backbone of damaged DNA molecules, or in some cases, to synthesize new strands of DNA by joining together larger fragments. Like polymerases, ligases do not act spontaneously but only under certain conditions. With a few exceptions, ligases only act on double-stranded molecules, and only on one strand of a molecule at a time. The two ends to be joined must be bound adjacent to one another on the complementary strand. The ligase creates a bond between the two nucleotides on the ends of the two strands, forming one contiguous piece of DNA. The ligase used in the OLA joins together two oligodeoxyribonucletides, or oligos (short single stranded segments of DNA), that are bound on adjacent sites to a perfectly complementary template; the presence of a mismatch between the template and the two oligos to be joined together will prevent ligation.
The oligos for an OLA are designed so that the juxtaposition of the adjacent ends occurs at a previously identified variable nucleotide on the template. If both of the oligos are completely complementary to the target sequence, including the variable nucleotide, then ligation will occur (Fig. 1). Different oligos are designed for each of the variant forms of the template so that all genotypes (heterozygotes as well as homozygotes) can be positively identified (Fig. 2). One oligo is designed so that it can be chemically captured to a fixed surface (e.g., the bottom of a 96-well plate). This oligo is referred to as the "hook." The second oligo is designed so that it can be detected using a colorimetric assay. This oligo is referred to as the "reporter." The DNA to be assayed is mixed together with the two oligos in the presence of the ligase, and then transferred to a 96-well plate capable of binding the hook (Fig 3). Unbound DNA and oligos are washed away: if the match between template and oligos is perfect, ligation occurs, and a color response is observed because the reporter is bound to the plate via its ligation to the captured hook. If a mismatch exists between the template and the oligos, ligation does not occur, the reporter oligo is washed away, and no color results. A separate reaction is set up and run for each variant at a polymorphic site to confirm the absence/presence of alleles of each type, and the reactions for all of the variants are run side-by-side. Heterozygotes will show a color response in the reactions for two variants, whereas homozygotes will show a color response in only one of the reactions.
We used the OLA technique to look at chinook salmon from the Snake River system. The polymorphism for which we designed our assay was identified by a previous study where we sequenced the D-loop of the mitochondrial DNA in several individuals. It is a single nucleotide polymorphism where the target contains either an A or a C at a certain site close to the phenylalanine t-RNA gene. The mitochondrial DNA is a haploid genome, so, in this case, there were no heterozygotes and the color response occurred in only one well for each individual.
We looked at individuals taken from Sawtooth Hatchery on the south fork of the Salmon River, from two different sites in the Upper Salmon River; and from Catherine Creek in the Grande Ronde drainage. Figure 4 depicts how the samples were arranged in this assay. In all pairs of wells, color in the left well indicates an A at the polymorphic site and color in the right well indicates a C. There are several things to note. First, the first two pairs of columns represent individuals from Sawtooth Hatchery. The individuals in the first pair of columns were sampled in 1990, and the individuals in the second pair were sampled in 1991. Though the sample size of eight individuals per population is much too small to draw any statistically significant conclusions, there is an apparent shift in the frequency of the A-type between collections from different years. The third and fourth pairs of wells represent populations from the Upper Salmon River. These were fish collected in 1991 from sites above Sawtooth Hatchery. The site "3 BRB" is just upstream from the hatchery, and "FC" designates Frenchman's Creek, a site about 20 miles upstream from the hatchery. The A-type is totally absent from these sites. The last two pairs of wells are individuals collected from Catherine Creek in 1990 and 1992. In both cases, we only had six individuals, so the bottom two pairs of wells are empty. Again, there is a difference in frequency between the sample collections from different years. Catherine Creek was often stocked with fish from other drainages, and there is a possibility that the frequency shifts between years are the result of different stocks returning, but that remains unresolved at this point.
In conjunction with PCR, the oligonucleotide ligation assay can be used to nonlethally screen juveniles as well as adults from endangered populations, or to gather historical data from archived samples of scales or otoliths. The assay requires prior knowledge of the exact location of the genetic variation to be assayed. At this point in time, most research groups are still attempting to identify variable markers, and the OLA is not appropriate for this application. However, when enough suitable markers have been identified, the OLA can be used to genotype thousands of individuals in a matter of days, and generation of the genetic baseline critical to the interpretation of population-level data will be possible in a time frame that is more amenable to many of the issues facing fisheries today.
Nickerson, D. A., R. Kaiser, S. Lappin, J. Stewart, L. Hood, and U. Landegren. 1990. Automated DNA diagnostics using ELISA-based oligonucleotide ligation assay. Proc. Natl. Acad. Sci. USA 87:8923-8927.
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