Bare Nanocapillary for DNA Separation and Genotyping Analysis in Gel-Free Solutions without Application of External Electric Field

• Published in: Analytical chemistry (Washington) 2008-07, Vol.80 (14), p.5583-5589
• Main Authors: Wang, Xiayan, Wang, Shili, Veerappan, Vijaykumar, Byun, Chang Kyu, Nguyen, Han, Gendhar, Brina, Allen, Randy D, Liu, Shaorong
• Format: Article
• Language: English
• Subjects: Analytical biochemistry: general aspects, technics, instrumentation; Analytical chemistry; Analytical, structural and metabolic biochemistry; Arabidopsis; Arabidopsis - genetics; Biological and medical sciences; Biotechnology; Buffers; Chemistry; Chromatographic methods and physical methods associated with chromatography; Chromatography; Deoxyribonucleic acid; DNA; DNA, Plant - genetics; DNA, Plant - isolation & purification; DNA, Single-Stranded - genetics; DNA, Single-Stranded - isolation & purification; Electric fields; Electrons; Electrophoresis; Exact sciences and technology; Fundamental and applied biological sciences. Psychology; Gels; Genetic engineering; Genetic technics; Genotype; Genotype & phenotype; In vitro gene amplification. Pcr technique; Methods. Procedures. Technologies; Nanostructures - chemistry; Other chromatographic methods; Pressure; Solutions
• ISSN: 0003-2700; 1520-6882
• DOI: 10.1021/ac800549k

In this work, we demonstrate DNA separation and genotyping analysis in gel-free solutions using a nanocapillary under pressure-driven conditions without application of an external electric field. The nanocapillary is a ∼50-cm-long and 500-nm-radius bare fused-silica capillary. After a DNA sample is injected, the analytes are eluted out in a chromatographic separation format. The elution order of DNA molecules follows strictly with their sizes, with the longer DNA being eluted out faster than the shorter ones. High resolutions are obtained for both short (a few bases) and long (tens of thousands of base pairs) DNA fragments. Effects of key experimental parameters, such as eluent composition and elution pressure, on separation efficiency and resolution are investigated. We also apply this technique for DNA separations of real-world genotyping samples to demonstrate its feasibility in biological applications. PCR products (without any purification) amplified from Arabidopsis plant genomic DNA crude preparations are directly injected into the nanocapillary, and PCR-amplified DNA fragments are well resolved, allowing for unambiguous identification of samples from heterozygous and homozygous individuals. Since the capillaries used to conduct the separations are uncoated, column lifetime is virtually unlimited. The only material that is consumed in these assays is the eluent, and hence, the operation cost is low.