A comparison of different lysis buffers to assess allele dropout from single cells for preimplantation genetic diagnosis

Single cell polymerase chain reaction (PCR) for preimplantation genetic diagnosis (PGD) requires high efficiency and accuracy. Allele dropout (ADO), the random amplification failure of one of the two parental alleles, remains the most significant problem in PCR-based PGD testing since it can result in serious misdiagnosis for compound heterozygous or autosomal dominant conditions. A number of different strategies (including the use of lysis buffers to break down the cell and make the DNA accessible) have been employed to combat ADO with varying degrees of success, yet there is still no consensus among PGD centres over which lysis buffer should be used (ESHRE PGD Consortium, 1999 ). To address this issue, PCR amplification of three genes (CFTR, LAMA3 and PKP1) at different chromosomal loci was investigated. Single lymphocytes from individuals heterozygous for mutations within each of the three genes were collected and lysed in either alkaline lysis buffer (ALB) or proteinase K/SDS lysis buffer (PK). PCR amplification efficiencies were comparable between alkaline lysis and proteinase K lysis for PCR products spanning each of the three mutated loci (ΔF508 in CFTR 90% vs 88%; R650X in LAMA3 82% vs 78%; and Y71X in PKP1 91% vs 87%). While there was no appreciable difference between ADO rates between the two lysis buffers for the LAMA3 PCR product (25% vs 26%), there were significant differences in ADO rates between ALB and PK for the CFTR PCR product (0% vs 23%) and the PKP1 PCR product (8% vs 56%). Based on these results, we are currently using ALB in preference to PK/SDS buffer for the lysis of cells in clinical PGD. Copyright © 2001 John Wiley & Sons, Ltd.     

THE INFLUENCE OF SETTINGS AND ENVIRONMENTAL EVENTS ON METAMOTIVATIONAL STATE AND AROUSAL: FURTHER TESTS OF REVERSAL THEORY ON ASIAN SAMPLES

The two studies reported further test reversal theory hypotheses concerning human psychological responses to different types of settings and environmental events on an Asian sample. In study 1, individuals in four settings representing different combinations of metamotivational state and arousal level (telic and paratelic/low arousal; telic and paratelic/high arousal) completed the telic state measure (TSM; Svebak & Murgatroyd, 1985). Significant differences between participants' mean scores across the four settings were obtained for TSM items serious–playful, planning–spontaneous, felt arousal, preferred arousal, effort and arousal discrepancy. In study 2 different environmental events in the same setting (changes in reading tasks), used in a crossover design, were used to induce reversals in individuals' operative metamotivational states. The results obtained from TSM scores pretask 1, between tasks and posttask 2. indicated significant time-of-testing interaction effects on the TSM serious–playful, planning–spontaneous and effort items. In addition, a main effect for TSM felt arousal was found. These results showed that reversals in metamotivational states were induced by the environmental event and that felt arousal scores increased across the three administrations of the TSM.     

The effects of job demands and control on employee attendance and satisfaction

We examined the impact of stressful job demands on employee attitudes and attendance. Using Karasek's (1979) theory of job decision latitude as the conceptual foundation, we hypothesized that mental and physical work demands would interact with employee beliefs of personal control. Survey data from 90 male manufacturing employees regarding their control beliefs were combined with objective job analysis data concerning mental and physical demands and one year's worth of archival data regarding unexcused absences, sick days, and days tardy. There were significant interactions between control and objective psychological demands that indicated that these demands were associated with higher levels of tardiness and sick days only under conditions of low perceived control. In contrast, subjective workload ratings showed no relationship with tardiness and sick days, but, in interaction with control, predicted work satisfaction and voluntary absence. We discussed these results in terms of a stress process that affects health-related attendance independent of employee attitudes.