Fundamental studies of DNA attachment and hybridization at a solid-liquid interface / Lynn Anne Sanguedolce.

Format:

• Book
• Manuscript
• Microformat
• Thesis/Dissertation

Author/Creator:

• Sanguedolce, Lynn Anne.

Contributor:

• Graves, David J., advisor.
• University of Pennsylvania.

Language:

English

Subjects (All):

• Penn dissertations--Chemical engineering.
• Chemical engineering--Penn dissertations.

Local Subjects:

• Penn dissertations--Chemical engineering.
• Chemical engineering--Penn dissertations.

Physical Description:

xv, 176 pages : illustrations ; 29 cm

Production:

1997.

Summary:

The merging of semiconductor technology with molecular biology has inspired DNA microarray development. The advantages of these devices include reduction of sample and reagent requirements and analysis time. The work in this thesis focuses on investigating DNA attachment and hybridization at the solid-liquid interface in a DNA microarray. Methods were developed to investigate the attachment of fluorescently-labeled, amino-modified oligonucleotides probes to surfaces modified with either 1,4-phenylene diisothiocyanate or glutaraldehyde. Hybridization of fluorescently-labeled target molecules, 15 nt and 219 nt, was investigated. Fluorescent intensities were imaged with a cooled charge-coupled device mounted on an epifluorescent microscope. Fluorescent intensity was not always proportional to hybridization or attachment levels as a result of fluorescent quenching, indicating analysis of fluorescent data must be interpreted within a range where fluorescent quenching does not occur in order not to underestimate the degree of attachment or hybridization. Discrimination between perfectly matched hybrids and mismatched hybrids was possible for single-base mismatches near the center of the probe. The effect of spacer molecule length was also studied. A 3-fold enhancement in hybridization was found for an increase in polyethylene glycol spacer length from 0-54 atoms. Hybridization intensity was correlated with CC content, dimer formation, duplex stability, melting and dissociation temperature for a set of sixteen oligonucleotides that span a region in the $\beta$-globin gene. Dimer formation was the only property that strongly correlated with hybridization intensity. Poor hybridization results were achieved with oligonucleotides that had the ability to form dimers with $>$50% of their sequence. Relative probe surface density was determined by staining with a ssDNA binding dye and the degree of attachment correlated with the degree of hybridization for fourteen of the sixteen probes. Hybridization intensity was weakly correlated with the predicted secondary structure of the target, suggesting that the analysis may not be robust enough or secondary structure of ssDNA fragments plays a minor role in predicting hybridization intensity. The lack of strong correlation between hybridization intensity and oligonucleotide sequence characteristics or target secondary structure indicates that reactions at the solid-liquid interface in DNA microarrays are a complex function of several parameters.

Notes:

• Supervisor: David J. Graves.
• Thesis (Ph.D. in Chemical Engineering) -- University of Pennsylvania, 1997.
• Includes bibliographical references.

Local Notes:

University Microfilms order no.: 98-14910.

OCLC:

187457658