Heteropolymeric Triplex-Based Genomic Assay® to Detect Pathogens or Single-Nucleotide Polymorphisms in Human Genomic Samples

Human genomic samples are complex and are considered difficult to assay directly without denaturation or PCR amplification. We report the use of a base-specific heteropolymeric triplex, formed by native duplex genomic target and an oligonucleotide third strand probe, to assay for low copy pathogen genomes present in a sample also containing human genomic duplex DNA, or to assay human genomic duplex DNA for Single Nucleotide Polymorphisms (SNP), without PCR amplification. Wild-type and mutant probes are used to identify triplexes containing FVL G1691A, MTHFR C677T and CFTR mutations. The specific triplex structure forms rapidly at room temperature in solution and may be detected without a separation step. YOYO-1, a fluorescent bis-intercalator, promotes and signals the formation of the specific triplex. Genomic duplexes may be assayed homogeneously with single base pair resolution. The specific triple-stranded structures of the assay may approximate homologous recombination intermediates, which various models suggest may form in either the major or minor groove of the duplex. The bases of the stable duplex target are rendered specifically reactive to the bases of the probe because of the activity of intercalated YOYO-1, which is known to decondense duplex locally 1.3 fold. This may approximate the local decondensation effected by recombination proteins such as RecA in vivo. Our assay, while involving triplex formation, is sui generis, as it is not homopurine sequence-dependent, as are “canonical triplexes”. Rather, the base pair-specific heteropolymeric triplex of the assay is conformation-dependent. The highly sensitive diagnostic assay we present allows for the direct detection of base sequence in genomic duplex samples, including those containing human genomic duplex DNA, thereby bypassing the inherent problems and cost associated with conventional PCR based diagnostic assays

Turning Points to Becoming a Tobacco Smoker: Smoking Initiation and Identity Change among Chinese Youth

In this study, we analyzed identity construction among young smokers in China, with three interconnected objectives: to theorize the turning points and career trajectory of smoking initiation; to account for their characteristics with interactionist processes; and to critically evaluate the applicability of classic typologies of identity change by Becker and Strauss. In-depth interviews with 24 late adolescents (ages 18–19) revealed a smoking initiation career path of four interconnected turning points, each characterized by interactionist processes. Smoker peers played a key role in facilitating overall career progression, and shame avoidance was crucial to their social dynamics. We also conclude that classic studies of turning points in general, and substance use specifically, are sufficiently broad and flexible to elucidate tobacco smoker identity construction in China, and facilitate a comparison of commonality and divergence among different “becoming” identities. The implications of these findings for tobacco control in China are discussed.</p