Assessing genetic polymorphisms using DNA extracted from cells present in saliva samples

<p>Abstract</p> <p>Background</p> <p>Technical advances following the Human Genome Project revealed that high-quality and -quantity DNA may be obtained from whole saliva samples. However, usability of previously collected samples and the effects of environmental conditions on the samples during collection have not been assessed in detail. In five studies we document the effects of sample volume, handling and storage conditions, type of collection device, and oral sampling location, on quantity, quality, and genetic assessment of DNA extracted from cells present in saliva.</p> <p>Methods</p> <p>Saliva samples were collected from ten adults in each study. Saliva volumes from .10-1.0 ml, different saliva collection devices, sampling locations in the mouth, room temperature storage, and multiple freeze-thaw cycles were tested. One representative single nucleotide polymorphism (SNP) in the catechol-<it>0</it>-methyltransferase gene (COMT rs4680) and one representative variable number of tandem repeats (VNTR) in the serotonin transporter gene (5-HTTLPR: serotonin transporter linked polymorphic region) were selected for genetic analyses.</p> <p>Results</p> <p>The smallest tested whole saliva volume of .10 ml yielded, on average, 1.43 ± .77 μg DNA and gave accurate genotype calls in both genetic analyses. The usage of collection devices reduced the amount of DNA extracted from the saliva filtrates compared to the whole saliva sample, as 54-92% of the DNA was retained on the device. An "adhered cell" extraction enabled recovery of this DNA and provided good quality and quantity DNA. The DNA from both the saliva filtrates and the adhered cell recovery provided accurate genotype calls. The effects of storage at room temperature (up to 5 days), repeated freeze-thaw cycles (up to 6 cycles), and oral sampling location on DNA extraction and on genetic analysis from saliva were negligible.</p> <p>Conclusions</p> <p>Whole saliva samples with volumes of at least .10 ml were sufficient to extract good quality and quantity DNA. Using 10 ng of DNA per genotyping reaction, the obtained samples can be used for more than one hundred candidate gene assays. When saliva is collected with an absorbent device, most of the nucleic acid content remains in the device, therefore it is advisable to collect the device separately for later genetic analyses.</p

Physiological responses to naturally occurring stressors: The roles of context and dehydroepiandrosterone

While HPA-axis and sympathetic responding to acute stress is an adaptive reaction, chronic activation can lead to the development of health problems. How both systems coordinate remains unclear, although recent reports on their asymmetry suggest independent responding. Hyperactive and asymmetrical acute response patterns are characteristic of physiological dysfunction; both are predictive of health problems. Acute physiological response variability is the product of interactions between individual differences and environmental contexts. The current research examined the effects of context on acute physiological responding in relation to DHEA and behavior. Study 1 examined exam-taking in college students. Exam stress significantly increased cortisol responding and increased negative affect compared to attending a lecture. Study 2, a replication of Study 1, found increased cortisol and negative affect on the exam day. Those whose cortisol increased the most tended to have low levels of DHEA, but those whose cortisol change was minimal tended to have higher levels of DHEA. Study 3 assessed cortisol and sympathetic (sAA) responding in hockey players during practice, game and rest days. Game day cortisol levels were higher compared to both practice and rest day levels, while sAA levels did not vary between days. Perceived stress and negative affect both predicted sAA reactivity on the game day. Study 4 found sAA increases, but cortisol decreases, following a sprinting activity in football players. DHEA reactivity was shown to mediate both sAA and cortisol responses, and behavioral traits of aggression and neuroticism correlated with both sAA and cortisol measures. Finally, Study 5 found no differences in cortisol or sAA levels between concert and rehearsal days in singers. Cortisol levels decreased on both days, however, following singing. DHEA and cortisol reactivity were correlated on the rehearsal day only, while traits of aggression and neuroticism were correlated with sAA and cortisol measures on both days. The current research increases our understanding of physiological stress responses by providing important links between contexts of stress, DHEA and behavior