|[Frontiers in Bioscience 1, c1-3, January 1, 1996]|
Solid phase purification and SSCP analysis of amplified genomic DNA by capillary electrophoresis
Silvana Debernardi, Massimo Luzzana and Gianluca De Bellis
Consiglio Nazionale delle Ricerche, Istituto di Tecnologie Biomediche Avanzate, L.I.T.A., Via Fratelli Cervi 93, 20090 Segrate, ITALY
Results and Discussion
The aim of the procedure reported here is to provide a protocol for SSCP analysis by capillary electrophoresis simplifying the interpretation of the results. To achieve this goal we propose to amplify the target genomic region using a biotinylated primer. The resulting product could be purified from buffer, dNTPs and primers by a magnetic solid phase procedure. Then, single stranded DNA could be rescued and analyzed by SSCP on a capillary electrophoresis apparatus running at a precise temperature avoiding renaturation problems and interference from other PCR components (7). Positive samples could be sequenced using the immobilized DNA strand (5).
In order to validate this hypothesis, we analyzed one sample that contained a heterozygous point mutation in the ß globin gene (Hb Abruzzo (6)). A normal sample was used as a control.
We expected the presence of two peaks for the heterozygous sample and one peak for the normal sample. Separation was optimized by using two capillary tubes of different lengths and by varying the voltage of the runs. We also analyzed different concentrations of non-crosslinked gels made by methylcellulose. We tested glycerol as a possible additive (0-10%). However, we did not note any benefical effect on separation (data not shown). Figure 1 (a-d) shows results obtained on a 36 cm capillary tube. Separation improved by decreasing voltage from 10 Kv to 4 Kv. This change resulted in widening of the peaks. At 2 Kv, no improvement in resolution was evident. Using a 50 cm capillary tube the same sample gave a good resolution at 8 Kv in about 25 minutes (Fig. 1 e).
Figure 1. CE electropherograms of ssDNA purified by magnetic beads.
The SSCP analysis was performed using a Hb Abruzzo sample. The sample was run in a 36 cm capillary tube at a) 10 Kv, b) 6 Kv, c) 4 Kv d) 2 Kv and e) 8 Kv in a 50 cm capillary tube. The two peaks represent the normal and the mutated DNA respectively.
The single stranded DNA, rescued as explained, was used for SSCP analysis on a non-denaturing polyacrylamide slab gel under standard conditions (8). Silver staining showed two bands for the heterozygous sample and a single band for the normal one (data not shown). These findings demonstrate the feasibility of this approach for conventional SSCP analysis.
Many technical factors affect the sensitivity of SSCP including the type of the gel matrix, temperature and conditions of electrophoresis(9). Therefore to identify all possible mutations it is necessary to test several experimental conditions where a given set of conditions could allow detection of only a part of mutations (1). Automated capillary electrophoresis systems allow testing all possible experimental parameters in a rapid and automated way. It also allows precise control of the temperature of the run, one of the most critical parameters in conformational analysis. Furthermore, CE confers several advantages over more conventional techniques such as fast separation and use of only minute quantities of sample. This technique also allows replacement of the non cross-linked polymer matrix by introducing a new aliquot after each run, thus, ensuring a high run-to-run reproducibility. The precise control of the running temperature is crucial to the reproducibility of the analysis and to the stability of the different conformations of single stranded polynucleotides. The use of solid phase purification facilitates data interpretation and offers the possibility to sequence the DNA attached to the magnetic beads to verify the presence of the screened mutations. The proposed approach is suitable both for large scale automated SSCP screening of known mutations after method optimization and for optimizing SSCP analysis of unknown mutations.