Date of Award

12-2011

Document Type

Campus Access Thesis

Degree Name

Master of Science (MS)

Department

Biotechnology and Biomedical Science

First Advisor

Maura Costello

Second Advisor

Richard Kesseli

Third Advisor

Linda Huang

Abstract

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.

Comments

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