DNA Synthesis, Purification, and Quantification
Faculty Mentor Name
Liang Xue
Research or Creativity Area
Natural Sciences
Abstract
DNA synthesis is an important technique used to produce readily available DNA oligonucleotides in a wide range of biochemistry experiments. In our laboratory, for instance, synthesized DNA strands are essential in studying molecular interactions with unique DNA structures, particularly DNA triplexes. DNA sequences were prepared via solid-phase DNA synthesis. In this automated process, the growing DNA oligonucleotide chain is anchored to a solid support (resin column) at its 3′ end and assembled one base at a time in the 3′→5′ direction. Because the strand stays attached to the column throughout the process, excess reagents and byproducts can be easily washed away with solvent between each coupling step. This approach significantly simplifies purification and reduces handling time. Following synthesis, the crude DNA products were purified utilizing denaturing polyacrylamide gel electrophoresis (gel purification). This technique separates DNA strands according to their length. We first prepared the gel and running buffers, loaded the samples, applied an electric field to migrate the DNA through the gel, and finally separated the bands from the gel. The purified DNA was recovered from the gel slices, yielding high-purity oligonucleotides suitable for further experiments. In the present work, three analytical techniques were employed to characterize the synthesized DNA: Matrix-Assisted Laser Desorption/Ionization (MALDI) and UV-Vis spectroscopy. MALDI-MS confirmed that the measured mass matched the theoretical molecular weight, thereby verifying the strand identity. UV-Vis spectroscopy was used to determine DNA yield from the preceding synthesis steps and to evaluate the efficiency of the synthesis. In this presentation, the synthesis and characterization of DNA oligonucleotides will be presented.
DNA Synthesis, Purification, and Quantification
DNA synthesis is an important technique used to produce readily available DNA oligonucleotides in a wide range of biochemistry experiments. In our laboratory, for instance, synthesized DNA strands are essential in studying molecular interactions with unique DNA structures, particularly DNA triplexes. DNA sequences were prepared via solid-phase DNA synthesis. In this automated process, the growing DNA oligonucleotide chain is anchored to a solid support (resin column) at its 3′ end and assembled one base at a time in the 3′→5′ direction. Because the strand stays attached to the column throughout the process, excess reagents and byproducts can be easily washed away with solvent between each coupling step. This approach significantly simplifies purification and reduces handling time. Following synthesis, the crude DNA products were purified utilizing denaturing polyacrylamide gel electrophoresis (gel purification). This technique separates DNA strands according to their length. We first prepared the gel and running buffers, loaded the samples, applied an electric field to migrate the DNA through the gel, and finally separated the bands from the gel. The purified DNA was recovered from the gel slices, yielding high-purity oligonucleotides suitable for further experiments. In the present work, three analytical techniques were employed to characterize the synthesized DNA: Matrix-Assisted Laser Desorption/Ionization (MALDI) and UV-Vis spectroscopy. MALDI-MS confirmed that the measured mass matched the theoretical molecular weight, thereby verifying the strand identity. UV-Vis spectroscopy was used to determine DNA yield from the preceding synthesis steps and to evaluate the efficiency of the synthesis. In this presentation, the synthesis and characterization of DNA oligonucleotides will be presented.
