Sequence-dependent separation of DNA fragments by capillary electrophoresis in the presence of SYBR Green I.

Detection of alterations in nucleotide sequences enables us to associate a phenotype with the underlying genetic information. Nucleotide sequencing is generally accepted as the ultimate method for this purpose. Alternative convenient methods have been developed for detection of polymorphisms or variations in nucleotide sequences, including single-strand conformation polymorphism (SSCP) analysis, denaturing gradient gel electrophoresis (DGGE) and temperature gradient gel electrophoresis (TGGE). These methods have been used for prognosis and diagnosis of cancers and genetic diseases and for predicting drug sensitivity of microorganisms. Because capillary electrophoresis (CE) in conjunction with a laser-induced fluorescence (LIF) detector can analyze DNA molecules in real time with higher sensitivity (8,9, 11), CE has been used for many applications of DNA analysis (6). For example, CE combined with the TGGE method detected point mutations (3). It was also suggested that CE-LIF detection has the potential to resolve identically sized DNA fragments (4,7). We examined the conditions for high-resolution separation of doublestranded (ds)DNA by CE-LIF (P/ACE System 5010 with a LIF detector and a 47-cm-long capillary; Beckman Instruments, Fullerton, CA, USA) and analyzed the effect of nucleotide composition on CE migration. Because intercalators increase sensitivity and/or resolution in CE (8,11), we systematically evaluated the effects of several intercalators (SYBR Green I, [Molecular Probes, Eugene, OR, USA], thiazole orange, acridine orange and acridine yellow) on the separation of pBR322 MspI digests, which include the same-size fragments with different sequences. Intercalators were added to the gel buffer (eCAP dsDNA 1000 Kit; Beckman Instruments) for the highest sensitivity (11). SYBR Green I provided the best resolution (data not shown). SYBR Green I concentrations of 0.25, 0.5, 1.0 and 2.5 μM were compared; 1.0 μM afforded the highest resolution. For sample loads of approximately 60 nL, less than 1 μg/mL of sample DNA gave the best resolution. Separation of dsDNA was better at 15° than at 25°C. The pressure injection mode and the internal standard in the kit were used to compare migration times precisely (1). Under the conditions, the difference in migration times of internal standard in every electrophoresis was less than 0.2% (data not shown). To identify what kind of sequence element(s) influence the CE separation, migration time and peak width of nine 90-bp DNA fragments obtained by polymerase chain reaction (PCR) from different sources were compared under the optimized conditions. These fragments are identical in length but showed differences in migration time and peak width on the CE-LIF detector (Figure 1), whereas they could not be separated into different peaks by electrophoresis in slab-type 10% polyacrylamide gels (data not shown). There was no relationship to differences in AT content. We defined any 4-bp sequence consisting entirely of either AT or GC residues as a “stretch” and examined the relationship between migration differences and the numbers of stretches. The presence of multiple stretches delayed migration and broadened a peak irrespective of whether the stretches consisted of either AT or GC (Figure 1). It is possible that the PCR amplification procedure affected the conformation of the 90-bp fragments. To exclude this possibility, DNA fragments obtained by digestion with restriction enzymes were used in the following experiment. A fission yeast gene mei2 is essential for meiosis, and the mutations mei2-644A and mei2-644Y645V abolish meiosis at a nonpermissive temperature (10). These mutations consist of