Donald Liu

Type

Text

Type

Thesis

Advisor

Rafailovich, Miriam H | Sokolov, Jonathan C | Meng, Yizhi

Date

2017-12-01

Keywords

Materials science | DNA Sequencing | Biophysics | Engineering | Bioengineering | Materials Science | Next Generation Sequencing | Soft Lithography | Surfaces

Department

Department of Materials Science and Engineering

Language

en_US

Source

This work is sponsored by the Stony Brook University Graduate School in compliance with the requirements for completion of degree.

Identifier

http://hdl.handle.net/11401/78274

Publisher

The Graduate School, Stony Brook University: Stony Brook, NY.

Format

application/pdf

Abstract

The development of sequencing since the Human Genome Project has greatly advanced. Current Next Generation Sequencing (NGS) platforms can produce more cost-effective sequencing in a shorter amount of time by using massively parallel sequencers. However, current NGS platforms have limitations in that only DNA fragments of size 100-300 bases may be analyzed. When producing these fragments of DNA, the positional order is often lost due to random fragmentation. Additionally, with repeats, insertions, and deletions of the genome, it further complicates the construction of contigs. Although companies such as Pacific Biosciences have developed longer-read technologies which sequences longer fragments (10 kilobases), the contig assembly problem remains for complex genomes. In this study, a newly developed method of fragmenting DNA that maintains the order is used. The method involves coating the surface of a soft lithography Poly dimethylsiloxane (PDMS) stamp with Dnase I enzyme. The stamp was placed onto a Poly (methyl methacrylate) – coated silicon wafer that has stretched linearized DNA on the surface, thereby inducing fragmentation in a controlled manner. The previously used methods of coating the PDMS with Dnase I enzymes often had inconsistent coatings because it was manually applied. Experiments were conducted utilizing a PDMS stamp having micron-sized microwells dipped into and withdrawn from a DNase I enzyme solution using a computer-controlled stepping motor and linear stage. Through surface tension, this method allowed the DNase I enzyme to remain in the microwells. Thereafter, using fluorescence microscopy, it was demonstrated that the DNase I enzyme was deposited in the microwells and had successfully cut at the desired locations. | 48 pages

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