Sequence-based separation of single-stranded DNA in capillary zone electrophoresis

Abstract

This thesis explores further the mechanism for the sequence-based separation. First, the fluorescence tag attached to DNA was investigated as the cause of the sequenced-based separation. The same migration order was observed for labeled and unlabeled strands; this eliminates the label as the basis of the separation. There have been reports about how high ionic strength solution could affect the size and ion distributions around DNA. Because the separation principle of CZE is based on the charge-to-size ratio, the effective charge and size (conformation) of DNA were characterized by different biophysical techniques (dynamic light scattering, fluorescence anisotropy, small angle X-ray scattering). Results from these techniques show little difference in size as well as effective charge among different strands when taking the standard deviation into account. Even for strands that do show some differences in size or charge, the observed differences were not predictive of the migration order. Therefore, the effect of high ionic strength on the properties of DNA probably is sequence-independent.

Separation of DNA has wide applications in biology, human health, and forensics. For example, genome sequencing, genotyping, and metagenomic analysis of microbial communities have all relied on methods for DNA separation. Among the methods, gel electrophoresis has been one of the most popular ones that utilizes porous gel to separation DNA of different lengths. However, DNA of the same length sometimes needs to be distinguished to get sequence information. Existing methods for sequence-based separation usually require sufficient differences in conformation or differences in resistance to thermal or chemical denaturation. A simple approach to sequence-based separation of DNA that solely based on the sequence would be helpful. Previous work in our lab showed that single-stranded DNA (ssDNA) could be separated from it’s mutations with as few as one base substitution by using simple high concentration buffers, such as 0.14M K-PO4, in capillary zone electrophoresis (CZE) without any matrices. Efforts have been made to explain this phenomenon; however, the exact mechanism for this sequence-based separation was still unclear.

The difference in mobility of DNA might be due to the internal properties of DNA. High ionic strength buffer might just magnify the difference by increasing the migration time in the phosphate CZE system. The effect of ionic strength on electroosmotic flow (EOF) mobility and electrophoretic (EP) mobility of DNA was measured and showed that both EOF and EP will decrease with increasing ionic strength. However, EOF is more sensitive to ionic strength and will decrease more dramatically than EP. Since EOF is larger than EP in our system and they have opposite directions, the apparent velocity is EP subtracted from EOF. At high ionic strength, the difference between EOF and EP decreases, thus leads to a slow apparent velocity and a long migration time. To test the intrinsic differences among DNA strands, a DNA library was built to investigate the relationship between sequence and mobility. A regression model is built to relate sequences to mobility. Further optimization of the model will be necessary to account for more variables in the sequences, and thereby improve the predictive capacity of the model.