Sequence-Based Separation of Single-Stranded DNA by using Nucleotidesin Capillary Electrophoresis: Focus on Phosphate

摘要

DNA analysis has widespread applicability in biology, medicine, biotechnology and forensics. DNA separation by length is readily achieved using sieving gels in electrophoresis. Separation by sequence is less simple, generally requiring adequate differences in native or induced conformation or differences in thermal or chemical stability of the strands that are hybridized prior to measurement. We previously demonstrated separation of four single-stranded DNA 76-mers that differ by only a few A-G substitutions based solely on sequence using guanosine-5′-monophosphate (GMP) in the running buffer. We attributed separation to the unique self-assembly of GMP to form higher order structures. Here we examine an expanded set of 76-mers designed to probe the mechanism of the separation and effects of experimental conditions. We were surprised to find that other ribonucleotides achieved the similar separation to GMP, and that some separation was achieved using sodium phosphate instead of GMP. Potassium phosphate achieved almost as good separations as the ribonucleotides. This suggests that the separation medium provides a physicochemical environment for the DNA that effects strand migration in a sequence-selective manner. Further investigation is needed to determine whetherthe mechanism involves specific interactions between the phosphates and the DNAstrands or is a result of other properties of the separation medium. Phosphategenerally has been avoided in DNA separations by capillary gel electrophoresisbecause its high ionic strength exacerbates Joule heating. Our results suggestthat phosphate compounds should be examined for separation of DNA based onsequence.