Masters Thesis

Improved procedure for extraction and purification of high molecular weight DNA from Lilium pardalinum

Previous studies indicate that genome size is not an indicator of organismal complexity. Moreover, non-coding portions of the genome, including transposable element (TE) repeats contribute to genome obesity. It is not clear, however, if TE content and therefore genome obesity is advantageous in evolution. The family Liliaceae is ideally suited for this investigation, since its family members show a range of genome sizes as well as significantly obese genomes. Sequencing the genomes is the first step in addressing this question in Liliaceae. Typically shotgun sequencing is the method of choice for whole genome sequencing. However, this approach is not suitable for complex genomes with sequence redundancy, such as those in Liliaceae plants. In these cases, BAC library based sequencing rather than shotgun sequencing is the preferred. In order to obtain high molecular weight DNA required for BAC library preparation, whole nuclei extraction for DNA purification was used in previous attempts at creating BAC library for Fritillaria species, a plant with one of the largest known genomes, by researchers Gangavarapu, Jayakar, Mishra and Patel. Their efforts yielded mixed results at best and it was seen that the DNA purified from the procedure was insufficient in quality and quantity. This thesis research outlines two specific aims: (1) To develop an alternate protocol to extract and purify high molecular weight (HMW) DNA suited for ligation with BAC vector, from the leaf tissue of Lilium pardalinum, a species representative of the Liliaceae family. (2) To demonstrate through a flow cytometry assay that the whole nuclei based DNA extraction is not suitable for this purpose. Once the genome is successfully extracted and purified, the high molecular weight (HMW) DNA can then be used to create BAC clones for whole genome sequencing of L.pardalinum, as well as other downstream applications.

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