Reliable detection of low template DNA (LTDNA) is dependent on reproducible allelic peaks and use of appropriately set thresholds. Analyzing LTDNA at different thresholds than high template DNA samples is discouraged by some scientists [1]. Thus, enhancing LTDNA samples is preferred if thresholds set for high template DNA are maintained for LTDNA samples. Post-amplification purification with the use of silica columns increases peak heights of single source LTDNA, while maintaining heterozygote balances of the sample before purification. LTDNA samples are difficult to interpret due to stochastic effects: allelic dropout and heterozygote imbalance. Interpretation of LTDNA mixtures is even more complex due to allelic sharing between contributors and allelic masking by artifacts. To determine if post-amplification purification of DNA from two-person LTDNA mixtures can improve profile interpretation, variable concentrations and mixture ratios of saliva extracts were applied to Macherey-Nagel NucleoSpin® and Qiagen MinElute® columns.
The peak heights and heterozygote balance of two-person LTDNA mixtures were compared before and after purification with each silica column. Contributor proportion and heterozygote balance were not significantly affected by purification, and the peak heights of samples improved with use of either silica column. However, peak heights were higher in samples purified with Qiagen MinElute® columns. Given the higher peak heights, fewer dropouts occurred in samples purified with Qiagen MinElute® columns, but the occurrence of locus dropout reduced after purification with either column.
The performance of each silica column was characterized by comparing fold increase and their individual effect on the primer front. The fold increase of samples purified with Qiagen MinElute® columns was higher than samples purified with Macherey-Nagel NucleoSpin® columns, but replicate purifications of Macherey-Nagel NucleoSpin® columns were more precise than replicate purifications of Qiagen MinElute® columns. Samples purified with Macherey-Nagel NucleoSpin® columns removed more primers than samples purified with Qiagen MinElute® columns.
Post-amplification purification with silica columns is a useful investigative tool for LTDNA, especially for LTDNA mixtures. Both silica columns increased peak height, maintained contributor proportion, maintained heterozygote balance, and reduced primer front, but the degree of fold increase and reduction in primer front must be considered to facilitate the decision of which silica column to use for post-amplification purification of LTDNA. Due to its precision, Macherey-Nagel NucleoSpin® columns are ideal for LTDNA mixture samples while Qiagen MinElute® columns are ideal for single source LTDNA due to its ability to elevate peak heights.
| Identifer | oai:union.ndltd.org:bu.edu/oai:open.bu.edu:2144/15373 |
| Date | 12 March 2016 |
| Creators | Rajapakse, Thanna |
| Source Sets | Boston University |
| Language | en_US |
| Detected Language | English |
| Type | Thesis/Dissertation |
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