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dc.contributor.advisorKennedy, Martin Alexander
dc.contributor.authorJodczyk, Sarah
dc.date.available2011-12-01T22:50:40Z
dc.date.copyright2011
dc.identifier.citationJodczyk, S. (2011). Establishment and validation of a telomere length assay (Thesis, Bachelor of Biomedical Sciences with Honours). Retrieved from http://hdl.handle.net/10523/2056en
dc.identifier.urihttp://hdl.handle.net/10523/2056
dc.description.abstractTelomeres are specialised structures, consisting of tracts of simple DNA sequence repeats that cap the ends of chromosomes. These structures play a vital role in maintaining the integrity of the genome and prevent the loss of genetic material that resides near the ends of the chromosome. As cells age and divide, the length of telomeric DNA sequences reduces and the progressive shortening of telomeres eventually leads to cell senescence and death. At the whole organism level telomere length appears to reflect cumulative lifetime stresses, and in humans, telomere shortening is associated with reduced longevity, many disease states including cancers, cardiovascular disease, mental illness, cognitive function and other phenotypes. The primary goals of this thesis were to establish and validate a method for measuring average telomere length, then apply it to examine a large longitudinal birth cohort for whom a great deal of data has been gathered over a period of 30 years. The telomere length assay is known as the monochrome multiplex quantitative polymerase chain reaction (MMqPCR) assay (1). This method provides relative quantification of average telomere length in a genomic DNA sample, by measuring the number of telomere repeats (T) normalised to the single copy reference gene albumin (S), and expressed as a T/S ratio. Signficant technical issues were encountered during the establishment of the assay, and it was found that the choice of hot-start polymerase was essential to the success of the assay. This was because the design of the telomere primers (1) necessitated an overlap of 3bp on their 3’ terminus, which led to primer dimer formation with six of fifteen hot-start polymerases tested. Data acquisition and calculation of T/S ratios was also challenging, and required export of data from the real-time PCR platform used (Roche LightCycler LC480), because the machine software was unable to handle the required analyses. Once established, the reproducibility of the assay was tested and it was found that the assay variation was sufficiently low to enable the detection of differences in telomere length between individuals, using duplicate measures for each sample. Further validation was attempted using Southern blotting of genomic DNA for comparison, but the number of samples obtained was insufficient to validate the assay. As telomere length is known to decrease with age, the performance of the assay was tested by comparing telomere length for subjects of different ages (seven, 25-40 and 50 years). A difference in telomere length was found in the 50 year old age group compared to younger age groups (P=0.001, 0.017). In addition, mouse embryonic stem cell DNA, reported to have ultra-long telomeres, was also examined and these samples did show very long telomere length in both the MMqPCR assay and Southern Blot. The MMqPCR assay was then applied to the Christchurch Health and Development Study (CHDS) which is a prospective longitudinal health study of over 1000 New Zealanders that have been closely followed in intimate detail from birth until the present time. This cohort provides a novel opportunity to examine associations between telomere length and life stress in a longitudinal setting, which should provide superior outcomes to the more typical cross-sectional or retrospective studies that form the bulk of the telomere biomarker literature. Preliminary MMqPCR data from a subset of the CHDS samples (n=255) was generated. Attempts to test associations between general measures of stress and telomere length were underpowered due to the small initial sample set used and the complexity and heterogeneity of available stress measures. However, a significant association was observed between individuals with the shortest telomeres and the highest rates of dependence using the single stress measures of nicotine and alcohol dependence, and the collective measure of substance dependence (P=0.0124, 0.0362 and 0.0008 respectively). No significant difference in telomere length was found with the stress measure of illicit drug dependence, but this group is of a small size. With the observed variability seen in the subset of samples assayed from the CHDS cohort (T/S ratios of 0.04-10.48) and the associations made between individual stressors and telomere length, there appears to be great potential in the use of telomere length as a biomarker of stress.
dc.format.mimetypeapplication/pdf
dc.language.isoen
dc.titleEstablishment and validation of a telomere length assay
dc.typeThesis
dc.date.updated2011-11-29T21:47:42Z
thesis.degree.disciplinePathology
thesis.degree.nameBachelor of Biomedical Sciences with Honours
thesis.degree.grantorUniversity of Otago
thesis.degree.levelHonours
otago.openaccessOpen
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