Phylogenetic analysis has been widely used to test the a priori hypothesis of epidemiological clustering in suspected transmission chains of HIV-1. Among studies showing strong support for relatedness between HIV samples obtained from infected individuals, evidence for the direction of transmission between epidemiologically related pairs has been lacking. During transmission of HIV, a genetic bottleneck occurs, resulting in the paraphyly of source viruses with respect to those of the recipient. This paraphyly establishes the direction of transmission, from which the source can then be inferred. Here, we present methods and results from two criminal cases, State of Washington v Anthony Eugene Whitfield , case number 04-1-0617-5 (Superior Court of the State of Washington, Thurston County, 2004) and State of Texas v Philippe Padieu , case numbers 219-82276-07, 219-82277-07, 219-82278-07, 219-82279-07, 219-82280-07, and 219-82705-07 (219th Judicial District Court, Collin County, TX, 2009), which provided evidence that direction can be established from blinded case samples. The observed paraphyly from each case study led to the identification of an inferred source (i.e., index case), whose identity was revealed at trial to be that of the defendant.
Syntheses of a unique set of energy transfer dye labeled nucleoside triphosphates, compounds 1-3, are described. Attempts to prepare these compounds were only successful if the triphosphorylation reaction was performed before coupling the dye to the nucleobase, and not the other way around. Compounds were prepared as both the 2'-deoxy (a) and 2',3'-dideoxy- (b) forms. They feature progressively longer rigid conjugated linkers connecting the nucleobase and the hydroxyxanthone moiety. UV spectra of the parent nucleosides 12-14 show that as the length of the linker increases so does the absorption of the donor in the 320-330 nm region, but with relatively little red-shift of the maxima. Fluorescence spectra of the same compounds show that radiation in the 320-330 nm region results in predominant emission from the fluorescein. When the linker is irradiated at 320 nm, the only significant emission observed corresponds to the hydroxyxanthone part of the molecules at 520 nm; this corresponds to an effective Stokes' shift of 200 nm. As the absorption at 320-330 nm by the linker increases with length, so does the intensity of the fluorescein emission. A gel assay was used to gauge relative incorporation efficiencies of compounds 1-3, dTTP, ddTTP, and 6-TAMRA-ddTTP. Throughout, the thermostable polymerase TaqFS was used, as it is the one most widely applied in high throughput DNA sequencing. This assay showed that only compounds 3 were incorporated efficiently; these have the longest linkers. Of these, the 2'-deoxy nucleoside 3 a was incorporated and did not prevent the polymerase from extending the chain further. The 2',3'-dideoxy nucleoside 3 b was incorporated only about 430 times less efficiently than ddTTP under the same conditions, and caused chain termination. The implications of these studies on modified sequencing protocols are discussed.
We present an approach called pulsed multiline excitation (PME) for measurements of multicomponent, fluorescence species and demonstrate its application in capillary electrophoresis for DNA sequencing. To fully demonstrate the advantages of PME, a fluorescent dye set has been developed whose absorption maxima span virtually the entire visible spectrum. Unlike emission wavelength-dependent approaches for identifying fluorescent species, the removal of the spectral component in PME confers a number of advantages including higher and normalized signals from all dyes present in the assay, the elimination of spectral cross-talk between dyes, and higher signal collection efficiency. Base-calling is unambiguously determined once dye mobility corrections are made. These advantages translate into significantly enhanced signal quality as illustrated in the primary DNA sequencing data and provide a means for achieving accurate base-calling at lower reagent concentrations.
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