Graduate NIH Training in Biotechnology
Trainees
Jeffrey Savard
Project Description
Our work has focused on the solution-based separation of nucleic acid targets using peptide nucleic acids (PNA). A PNA is a DNA analog in which the negative phosphodiester backbone of DNA has been replaced by a neutral polyamide backbone. This neutrality confers greater stability to the PNA-DNA triplex, and offers greater mismatch sensitivity compared to traditional DNA hybridization. Our lab has modified the PNA by adding an aliphatic carbon tail, which we term Peptide Nucleic Acid Amphiphiles (PNAA). This addition confers significant hydrophobicity to PNA, and upon binding, to DNA as well. Hence, this tail allows us to sequence-specifically isolate and purify target DNA via separation techniques based on differences in sample hydrophobicity, such as hydrophobic interaction chromatography (HIC), or micellar electrokinetic chromatography (MEKC). We have shown facile separation of DNA oligomers and double-stranded DNA from complex mixtures using a column-based HIC approach, and have transferred this technology to a rapid, highly parallel plate-based assay. Significant work towards separation of extremely long (greater than 1000 bases) single-stranded DNA targets from PCR mixtures using MEKC has also been accomplished. Our next aim is to utilize this separation modality in the rapid and sequence-specific purification of RNA targets from cell lysates.
Courses
06607 Physical Chemistry of Colloids and Surfaces
06608 Engineering Safety
06621 Biotechnology and Environmental Processes
06702 Advanced Kinetics
06704 Advanced Heat and Mass Transfer
06703 Advanced Fluid Dynamics
06705 Advanced Chemical Thermodynamics
06713 Mathematical Techniques
BIOE2810 Biomaterials and Biocompatibility
Foundations of Biomedical Sciences Conference
Foundations of Biomedical Sciences Lecture
PT708 Intro to Molecular Cell Biology
Scientific Ethics
Publications
"Sequence-Specific Purification of DNA Oligomers in Hydrophobic Interaction Chromatography using Peptide Nucleic Acid Amphiphiles," J.M. Savard and J.W. Schneider, Biotech. Bioeng., DOI 10.1002/bit. 21242, (2006).
"Triplex Formation and Strand Invasion of Double-Stranded DNA with Peptide Nucleic Acid Amphiphiles for Sequence-Specific DNA Purification," J.M. Savard, J.P. Vernille, and J.W. Schneider, submitted to J. Chrom. B (2006).
"Hybridization of Multiple Peptide Nucleic Acid Amphiphiles for the Separation of Long DNA Targets in Micellar Electrokinetic Chromatography," J.M. Savard, S.T. Grosser and J.W. Schneider, submitted to J. Am. Chem. Soc. (2006).
"Specific DNA Purification using PNA Amphiphiles in Micellar Electrokinetic Chromatography," S.T. Grosser, J.M. Savard and J.W. Schneider, submitted to Electrophoresis (2006).
Presentations
"Adsorption of Hydrophobically Modified DNA to Micelles, Liposomes, and Microemulsions," S.T. Grosser, J.M. Savard, and J.W. Schneider, AIChE Annual Meeting, San Francisco, CA (2006).
"The Effect of Sense and Multiple PNAA Binding in Sequence-Specific DNA Separations using MEKC," J.M. Savard, S.T. Grosser, and J.W. Schneider, Carnegie Mellon ChEGSA Symposium, Pittsburgh, PA (2006).
"Back-Extraction of PNA Affinity Tags following HIC Purification of DNA Targets (poster)," J.M. Savard and J.W. Schneider, Frontiers in Peptide Nucleic Acid and Related Technologies Symposium, Pittsburgh, PA (2006).
"Back-Extraction of PNA Affinity Tags following HIC Purification of DNA Targets," J.M. Savard and J.W. Schneider, AIChE Annual Meeting, Cincinnati OH (2005).
"Back-Extraction of PNA Affinity Tags following HIC Purification of DNA Targets," J.M. Savard and J.W. Schneider, Carnegie Mellon ChEGSA Symposium, Pittsburgh, PA (2005).
"DNA Separations using Peptide Nucleic Acid Amphiphiles as Affinity Tags in Hydrophobic Interaction Chromatography," J.M. Savard and J.W. Schneider, 13th International Conference on Biopartitioning and Purification, Amsterdam Netherlands (2005).
"Affinity Ligands for Sequence-Specific Duplex DNA Separation in Hydrophobic Interaction Chromatography," J.W. Schneider, J.P. Vernille, and J. Savard, AIChE Annual Meeting, Austin TX (2004).
"DNA Enrichment in Hydrophobic Interaction Chromatography using Peptide Nucleic Acid Amphiphiles (poster)," J.M. Savard, J.P. Vernille, and J.W. Schneider, Carnegie Mellon ChEGSA Symposium, Pittsburgh, PA (2004).
Seminar Attendance
All Department of Chemical Engineering, CMU seminars
Select Department of Biomedical Engineering, CMU seminars
Journal club meetings
Advisor
James W. Schneider
Associate Professor of Chemical Engineering
Carnegie Mellon University
