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Comparative Study
. 2001 Jul 15;29(14):E71.
doi: 10.1093/nar/29.14.e71.

Combined SSCP/duplex analysis by capillary electrophoresis for more efficient mutation detection

P Kozlowski  1 , W J Krzyzosiak
Affiliations
Comparative Study

Combined SSCP/duplex analysis by capillary electrophoresis for more efficient mutation detection

P Kozlowski et al. Nucleic Acids Res. .

Abstract

SSCP and heteroduplex analysis (HA) continue to be the most popular methods of mutation detection due to their simplicity, high sensitivity and low cost. The advantages of these methods are most clearly visible when large genes, such as BRCA1 and BRCA2, are scanned for scattered unknown mutations and/or when a large number of DNA samples is screened for specific mutations. Here we describe a novel combined SSCP/duplex analysis adapted to the modern capillary electrophoresis (CE) system, which takes advantage of multicolor labeling of DNA fragments and laser-induced fluorescence detection. In developing this method, we first established the optimum conditions for homoduplex and heteroduplex analysis by CE. These were determined based on comprehensive analysis of representative Tamra-500 markers and BRCA1 fragments at different concentrations of sieving polymer and temperatures in the presence or absence of glycerol. The intrinsic features of DNA duplex structures are discussed in detail to explain differences in the migration rates between various types of duplexes. When combined SSCP/duplex analysis was carried out in single conditions, those found to be optimal for analysis of duplexes, all 31 BRCA1 and BRCA2 mutations, polymorphisms and variants tested were detected. It is worth noting that the panel of analyzed sequence variants was enriched in base substitutions, which are usually more difficult to detect. The sensitivity of mutation detection in the SSCP portion alone was 90%, and that in the duplex portion was 81% in the single conditions of electrophoresis. As is also shown here, the proposed combined SSCP/duplex analysis by CE has the potential of being applied to the analysis of pooled genomic DNA samples, and to multiplex analysis of amplicons from different gene fragments. These modifications may further reduce the costs of analysis, making the method attractive for large scale application in SNP scanning and screening.

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Figures

Figure 1
Figure 1
Migration rates of 100 bp (white bars) and 500 bp (black bars) fragments of Tamra 500 DNA size standard, as expressed in the number of scans, in different conditions of electrophoresis. (A) Temperature dependence (30–45°C) at a constant 5% GeneScan polymer concentration; (B) temperature dependence (30–65°C) at a constant 5% GeneScan polymer concentration containing 10% glycerol; (C) influence of different concentrations of GeneScan polymer (2–5%) containing 10% glycerol at a constant temperature (30°C).
Figure 2
Figure 2
DA of six sequence variants of the BRCA1 gene. The characteristics of samples analyzed in panels 1–4 are described in Results. Positions of homoduplexes of the more frequent polymorphic variant (f), less frequent polymorphic variant (r), as well as the positions of two heteroduplexes I and II are indicated above each set of electrophoregrams. Duplexes labeled with 6-carboxy-2′,7′-dimethoxy-4′,5′-dichlorofluorescein (JOE) and FAM are shown as dark and light peaks, respectively. DNA size standards are shown as white peaks.
Figure 3
Figure 3
Separations of duplexes of eight fragments of the BRCA1 gene in different conditions of electrophoresis. The graphs show the differences in migration rates between the homoduplex of the less frequent polymorphic variant (black bars), as well as the faster and slower migrating heteroduplexes (gray and white bars, respectively) relative to the homoduplex of the more frequent polymorphic variant. Bars above and below the x-axis indicate, respectively, duplexes migrating slower and faster than the homoduplex of the more frequent polymorphic variant. (A), (B) and (C) are denoted as in the legend to Figure 1. In the case of BRCA1 fragment 20 with 12 bp insertion, in which migration of heteroduplexes is much slower than that of homoduplexes, the number of scans representing the distance between two homoduplexes is indicated above the corresponding bars.
Figure 4
Figure 4
(Opposite and above) Examples of combined SSCP/DA of seven different fragments of the BRCA1 and BRCA2 genes. Left and right panels show in magnification the duplex and SSCP portions of electrophoregrams, respectively. In addition, in the inset to (A), the real distance between duplex and SSCP portions is shown in a continuous fragment of electrophoregram. The peaks drawn with black and gray lines represent strands labeled with FAM or Tamra and with JOE or 6-carboxy-X-rhodamine (ROX), respectively. The shaded peaks represent fragments of the DNA size standard. Electrophoregrams of BRCA1 fragments spanning common polymorphism sites are shown (A–C). The electrophoregram representing homozygote for the more frequent polymorphic variant is shown in the upper panel, that representing the homozygote for the less frequent polymorphic variant in the lower panel, and that of the heterozygote is displayed in the central panel. Electrophoregrams of gene fragments spanning mutation or rare sequence variant sites are shown (D–G). In each case, the electrophoregrams representing the wild-type sequence or the common sequence variant (upper panel) are shown along with electrophoregrams representing the heterozygous sample (lower panel). Genotypes of the analyzed mutations, polymorphisms and variants are specified in each electrophoregram.
Figure 4
Figure 4
(Opposite and above) Examples of combined SSCP/DA of seven different fragments of the BRCA1 and BRCA2 genes. Left and right panels show in magnification the duplex and SSCP portions of electrophoregrams, respectively. In addition, in the inset to (A), the real distance between duplex and SSCP portions is shown in a continuous fragment of electrophoregram. The peaks drawn with black and gray lines represent strands labeled with FAM or Tamra and with JOE or 6-carboxy-X-rhodamine (ROX), respectively. The shaded peaks represent fragments of the DNA size standard. Electrophoregrams of BRCA1 fragments spanning common polymorphism sites are shown (A–C). The electrophoregram representing homozygote for the more frequent polymorphic variant is shown in the upper panel, that representing the homozygote for the less frequent polymorphic variant in the lower panel, and that of the heterozygote is displayed in the central panel. Electrophoregrams of gene fragments spanning mutation or rare sequence variant sites are shown (D–G). In each case, the electrophoregrams representing the wild-type sequence or the common sequence variant (upper panel) are shown along with electrophoregrams representing the heterozygous sample (lower panel). Genotypes of the analyzed mutations, polymorphisms and variants are specified in each electrophoregram.
Figure 5
Figure 5
Modifications increasing throughout of combined SSCP/DA. (A) Multiplex analysis of three BRCA1 fragments: 11.22, 13 and 20. The electrophoregrams represent three possible combinations of genotypes in BRCA1 fragments 11.22, 13 and 20: 1, aa, aa, aa; 2, ab, ab, ab; and 3, bb, bb, ab, respectively. The symbols aa, ab and bb represent the more frequent homozygote, heterozygote and less frequent homozygote, respectively. (B) DNA pooling prior to PCR. 1, SSCP/duplex pattern of the BRCA1 5465 G/A heterozygous mutant; 2 and 3, the corresponding patterns of the mutant sample diluted, respectively, two and three times with DNA samples of the wild-type sequence 5465G; 4, SSCP/duplex pattern of DNA fragment with the pure wild-type sequence (5465G). Note that the peak shown by arrow distinguishing the mutant sequence pattern from that of the wild-type sequence contributes to 25% of the total duplex signal and to ∼50% of the signal produced by one of the conformers of the FAM-labeled strand (black line).
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