Melting curve analysis of SNPs (McSNP): a gel-free and inexpensive approach for SNP genotyping.

High-throughput methods for assaying DNA variation require two important steps: (i) discriminating the variation and (ii) detecting the signal. In this report, we describe a novel SNP genotyping method that we refer to as melting curve analysis of SNPs (McSNP). McSNP combines a classic approach for discriminating alleles, restriction enzyme digestion, with a more recent method for detecting DNA fragments, melting curve analysis. Melting curve analysis is performed by slowly heating DNA fragments in the presence of the dsDNA-specific fluorescent dye SYBR Green I. As the sample is heated, fluorescence rapidly decreases when the melting temperature of a particular fragment is reached. We show that it is possible to determine the composition of simple mixtures of DNA fragments, such as those that result from restriction enzyme digestions of short PCR products. McSNP is well suited for high-throughput genotyping because 96 samples can be analyzed and automatically scored in 20 min. Our results clearly demonstrate that McSNP is a simple, inexpensive, and accurate means of genotyping SNP variation.

Comparison of human apoA-I expression in mouse models of atherosclerosis after gene transfer using a second generation adenovirus.

Gene transfer and expression of apolipoprotein A-I (apoA-I), the major protein component of high density lipoproteins (HDL), is a potentially attractive method for investigating the effects of apoA-I on atherosclerosis. We constructed a second generation recombinant adenovirus encoding the human apoA-I cDNA. This adenoviral vector or a control vector was injected intravenously into apoE-deficient mice fed a chow diet and low density lipoprotein (LDL) receptor (LDLR)-deficient mice fed Western diet, as well as control wild-type C57BL/6 mice. The mean peak plasma human apoA-I concentrations were 235, 324, and 276 mg/dL in apoE-deficient, LDLR-deficient, and wild-type mice, respectively. Human apoA-I concentrations decreased rapidly in apoE-deficient mice and were barely detectable 6 weeks after injection. In contrast, substantially higher levels of human apoA-I were sustained in LDLR-deficient mice. In wild-type mice, human apoA-I levels decreased more rapidly than in LDLR-deficient mice, but could still be detected in plasma for up to 8 months after virus injection. In apoE-deficient mice a substantial fraction of human apoA-I was found associated with triglyceride (TG)-rich lipoproteins; in contrast, in LDLR-deficient and wild-type mice the majority of human apoA-I was found in the HDL fraction. Finally, expression of human apoA-I caused a transient but significant increase in triglyceride levels in all three mouse models. In summary: 1) a second generation recombinant adenovirus resulted in high-level expression of human apoA-I in mice; 2) significantly higher levels of human apoA-I persisted for a longer time in LDLR-deficient mice compared with apoE-deficient mice; and 3) substantial human apoA-I was found associated with TG-rich lipoproteins in apoE-deficient but not LDLR-deficient mice.

Population structure in admixed populations: effect of admixture dynamics on the pattern of linkage disequilibrium.

Gene flow between genetically distinct populations creates linkage disequilibrium (admixture linkage disequilibrium [ALD]) among all loci (linked and unlinked) that have different allele frequencies in the founding populations. We have explored the distribution of ALD by using computer simulation of two extreme models of admixture: the hybrid-isolation (HI) model, in which admixture occurs in a single generation, and the continuous-gene-flow (CGF) model, in which admixture occurs at a steady rate in every generation. Linkage disequilibrium patterns in African American population samples from Jackson, MS, and from coastal South Carolina resemble patterns observed in the simulated CGF populations, in two respects. First, significant association between two loci (FY and AT3) separated by 22 cM was detected in both samples. The retention of ALD over relatively large (>10 cM) chromosomal segments is characteristic of a CGF pattern of admixture but not of an HI pattern. Second, significant associations were also detected between many pairs of unlinked loci, as observed in the CGF simulation results but not in the simulated HI populations. Such a high rate of association between unlinked markers in these populations could result in false-positive linkage signals in an admixture-mapping study. However, we demonstrate that by conditioning on parental admixture, we can distinguish between true linkage and association resulting from shared ancestry. Therefore, populations with a CGF history of admixture not only are appropriate for admixture mapping but also have greater power for detection of linkage disequilibrium over large chromosomal regions than do populations that have experienced a pattern of admixture more similar to the HI model, if methods are employed that detect and adjust for disequilibrium caused by continuous admixture.

Ancestral proportions and admixture dynamics in geographically defined African Americans living in South Carolina.

We analyzed admixture in samples of six different African-American populations from South Carolina: Gullah-speaking Sea Islanders in coastal South Carolina, residents of four counties in the "Low Country" (Berkeley, Charleston, Colleton, and Dorchester), and persons living in the city of Columbia, located in central South Carolina. We used a battery of highly informative autosomal, mtDNA, and Y-chromosome markers. Two of the autosomal markers (FY and AT3) are linked and lie 22 cM apart on chromosome 1. The results of this study indicate, in accordance with previous historical, cultural, and anthropological evidence, a very low level of European admixture in the Gullah Sea Islanders (m = 3.5 +/- 0.9%). The proportion of European admixture is higher in the Low Country (m ranging between 9. 9 +/- 1.8% and 14.0 +/- 1.9%), and is highest in Columbia (m = 17.7 +/- 3.1%). A sex-biased European gene flow and a small Native American contribution to the African-American gene pool are also evident in these data. We studied the pattern of pairwise allelic associations between the FY locus and the nine other autosomal markers in our samples. In the combined sample from the Low Country (N = 548), a high level of linkage disequilibrium was observed between the linked markers, FY and AT3. Additionally, significant associations were also detected between FY and 4 of the 8 unlinked markers, suggesting the existence of significant genetic structure in this population. A continuous gene flow model of admixture could explain the observed pattern of genetic structure. A test conditioning on the overall admixture of each individual showed association of ancestry between the two linked markers (FY and AT3), but not between any of the unlinked markers, as theory predicts. Thus, even in the presence of genetic structure due to continuous gene flow or some other factor, it is possible to differentiate associations due to linkage from spurious associations due to genetic structure.