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Ultra-long Read Sequencing for Whole Genomic DNA Analysis

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A 502-Base Free-Solution Electrophoretic DNA Sequencing Method Using End-Attached Wormlike Micelles.

Stephen B Istivan¹, Daniel K Bishop¹, Angela L Jones¹

¹Department of Chemical Engineering and ‡Department of Biomedical Engineering, Carnegie Mellon University , Pittsburgh, Pennsylvania 15213, United States.

Analytical Chemistry

|October 13, 2015

Summary

Wormlike nonionic micelles significantly improve Sanger sequencing resolution up to 502 bases in end-labeled free-solution electrophoresis (ELFSE). This novel micelle-ELFSE method offers a nearly two-fold increase in read length and faster analysis times.

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Area of Science:

Analytical Chemistry
Biochemistry
Molecular Biology

Background:

End-labeled free-solution electrophoresis (ELFSE) is a technique for DNA sequencing.
Improving resolution and read length in ELFSE is crucial for accurate DNA analysis.

Purpose of the Study:

To investigate the use of wormlike nonionic micelles as drag-tags in ELFSE.
To enhance the single-base resolution and read length of Sanger sequencing products.

Main Methods:

Utilized wormlike nonionic micelles in running buffers for micelle-ELFSE.
Employed "CiEj" buffers with specific concentrations of C12E5, C10E5, and urea.
Analyzed Sanger sequencing products using capillary electrophoresis.

Main Results:

Achieved single-base resolution of Sanger sequencing products up to 502 bases, a nearly 2-fold improvement.
Demonstrated a runtime of 35 minutes for full product elution in a 40 cm capillary.
Showcased a combined 14-minute analysis using two different micelle buffers for the full range of lengths.

Conclusions:

Wormlike nonionic micelles effectively serve as drag-tags, enhancing ELFSE performance.
Resolution is primarily limited by diffusion and wall adsorption, not micelle polydispersity.
The developed micelle-ELFSE method offers a significant advancement in DNA sequencing analysis.