Fluorescence-based SSCP analysis with automatic allele detection demonstrated for the factor V Leiden mutation.
[FIGURE 1 OMITTED]
The primers used in PCR were described previously (2). For capillary electrophoresis, in PCR the forward and reverse primers were 5' fluorescently labelled with 6-carboxy-fluorescein (FAM) and 4,7,2',4',5',7'-hexachloro-ocarboxyfluorescein (HEX), respectively. To 1 [micro]L of the diluted PCR product (diluted 1:30 with HZO), 10.5 [micro]L of formamide, 0.5 [micro]L of a size marker (GeneScan-500 Tamra, PE Applied Biosystems), and 0.5 [micro]L of 0.3 mol/L NaOH were added, and the sample was denatured at 90 [degrees]C for 2 min. After the samples were chilled on ice, they were placed in the tray. Capillary electrophoresis was performed with the ABI Prism 310 Genetic Analyzer (PE Applied Biosystems) in a 50 g/L GeneScan[TM] polymer (PE Applied Biosystems) with 100 mL/L glycerol and 1 X Tris-borate-EDTA buffer (0.1 mol/L Tris, 0.077 mol/L boric acid, and 0.0025 mol/L EDTA, pH 8.5). A temperature of 30 [degrees]C and a running time of 30 min were chosen. In the case of the wild-type allele, two different peaks were found for both sense and antisense single strand. It is likely that more than one stable conformation was formed for each single strand. In the case of the mutation, additional peaks were detected (Fig. 1).
To perform an automatic evaluation of the mutation, an important point is the reproducibility of the complex peak pattern. The analysis of different electrophoresic runs over 15 days confirmed the constant appearance of the peak pattern. Characteristic peaks for the wild-type and mutant alleles were defined by their retention time and height. For the sense strand (FAM), the peaks were defined by the following data points: wild-type 1, 5756; wild-type 2, 5809; mutant 1, 5736; mutant 2, 5788, and for the antisense strand (HEX): wild-type, 5867; mutant, 5738. In the antisense strand, one wild-type and one mutant peak were not considered for automatic evaluation because the two peaks partially overlapped. The variation of the peak retention time was less than [+ or -]8 data points for the sense strand and less than [+ or -]20 data points for the antisense strand.
The retention time of the peaks and their variations were listed in the Genotyper[TM] analysis software (Version 2.0; PE Applied Biosystems), and a macro was created for the analysis of the data (clear table; clear labels; select blue lanes; and select green lanes; label category peaks with the category's name; set up table with one category and one lane per row; append rows to table; show the table window; set cell row 1 column 1 to sample name; show the plot window). In the diagram, the peaks were named wild-type or mutant, and in the resulting table the summary of analysis was presented (Fig. 1). In the table, the peaks were named wild-type for the sense and antisense single strands in the case of the homozygous presence of the wild-type allele. In the case of homozygosity of the mutation, only peaks defined as mutant were recognized; in the heterozygous state, a combination of both was present. In routine usage, the practicability and reliability of the evaluation program has been demonstrated.
The ABI 310 Genetic Analyzer has been widely distributed in clinical laboratories in the last 2 years. The main applications of this system are sequencing and fragment analysis. In our report, we showed that SSCP analysis could also be performed with high reliability by capillary electrophoresis. For the first time, the combination of SSCP analysis and automatic evaluation of the mutation has been shown, even in cases where a complex SSCP peak pattern was present. In the diagnostic DNA testing, this approach is advantageous in the analysis of small sample numbers (as little as one sample).
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Jurgen Geisel,* Tanja Walz, Marion Bodis, Sabine Quast, and Wolfgang Herrmann
(Klinisch-Chemisches Zentrallabor, Universitatskliniken des Saarlandes, Oskar-Orth-Strasse, 66421 Homburg/Saar, Germany; * author for correspondence: fax 6841 163109, e-mail firstname.lastname@example.org)
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|Title Annotation:||Technical Briefs|
|Author:||Geisel, Jurgen; Walz, Tanja; Bodis, Marion; Quast, Sabine; Herrmann, Wolfgang|
|Date:||Sep 1, 1998|
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