Date of Award
Campus Access Thesis
Master of Science (MS)
Biotechnology and Biomedical Science
Size selection creates a bottleneck in the high throughput Illumina library construction process. This study tested three alternative gel extraction methods to replace the conventional MinElute spin column, and with the ultimate goal of developing an automated protocol. New England Biolabs gel extraction method of β-Agarase enzyme/ethanol precipitation yielded lower DNA yield in comparison with MinElute spin column. When β-Agarase enzyme was coupled with SPRI magnetic beads, the yield was higher than the MinElute yield. However, Illumina sequencing metrics showed that this method compromised high GC content regions above 70% GC in human DNA. The most successful method evaluated was a novel gel extraction method termed QG-SPRI, which consists of dissolving gel excisions in QG buffer followed by SPRI purification. This method is amenable to automation and has resulted in sample yield and sequencing metrics that are as equal as or better than the MinElute spin column.
Bourzgui, Imane, "Alternative Gel Extraction Methods for High Throughput Illumina Sequencing Process" (2011). Graduate Masters Theses. 81.