Sequence-based separation of ssDNA in capillary electrophoresis

Abstract

DNA analysis has widespread applicability in biology, medicine, biotechnology and forensics. DNA separation by fragment length can be readily achieved using sieving gels in electrophoresis. Separation by sequence has not been as simple, generally requiring adequate differences in native or induced conformation between strands or differences in thermal or chemical stability of the strands that are hybridized prior to measurement. Previous work in the McGown group showed that four single-stranded DNA 76-mers that differ by only a few A-G substitutions could be separated based solely on sequence, simply by adding guanosine-5'-monophosphate disodium (GMP-Na2) to the running buffer. In this dissertation, further investigations of the separation conditions and mechanisms were studied.

A series of experiments were designed and performed to understand the separation mechanism. Ribonucleotides including adenosine monophosphate (AMP) and uridine monophosphate (UMP) achieved similar separation to GMP, which confirmed that the separation is not based on specific interactions between self-assembled GMP structures and the DNA. Similar separations also were achieved by using dGMP and GMP with glucose or ribose, indicating that the sugar ring is not the cause of the separation. Replacing GMP with other salts achieved various degrees of separation, and the separation by potassium phosphate was almost as good as GMP. We therefore concluded that the separation mechanism is most likely based on general salt effects.

In order to explore the separating ability of our technique, we examined an expanded set of ten different DNA 76-mers as a target mixture for separation. Under the optimized condition, the ten single-stranded DNAs (ssDNA) were separated into 7 peaks, within which four strands without secondary structures or G bases were separated into 3 peaks. In the course of these experiments, however, we discovered that the working hypothesis, which attributed the separation to the unique self-assembly of the GMP to form G-quadruplex structures, was incorrect.

To better understand the basis of the separation, we designed a set of ten ssDNA 15-mers that differ by only one or two bases and studied the separation under various conditions. Results demonstrate that the separation is influenced by the particular cations and anions in the salt as well as temperature and pH. Experiments to probe adsorption of the DNA to the inner capillary walls and circular dichroism (CD) spectroscopy to probe the structures of the 15-mers were performed. The results indicate that high salt concentration could impact DNA interactions with the inner capillary surface, and that the 15-mers have no well-defined secondary structure, which indicates that the separation is based on sequence alone. This work is a new approach to sequence-based separation of same-length ssDNA that in combination with length-based separation, will provide a powerful tool for analysis of DNA especially in complex samples such as forensic samples, microbial communities and biofilms. Its applicability also extends to new, simple and low cost devices for health care.