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Fig. 5. Fragment-size analysis using CE and fluorescence detection: spinocerebellar ataxia type I. The electopherograms show PCR products using primers flanking the CAG repeat of the ataxin-1 gene. The forward PCR primer is labeled with 6-FAM; the resultant peaks are shown in blue. The PCR products are mixed with a ROX-labeled size marker, shown in red, and coinjected for capillary electrophoresis on an ABI 3100 (Applied Biosystems, Foster City, CA). (A) a patient with 2 normal alleles, 30 and 36 repeats (222 and 239 bases, respectively; (B) a patient with normal allele of 26 repeats (211 bases) and 1 expanded allele of 68 repeats (332 bases). This expansion is diagnostic of spinocerebellar ataxia type I. (Courtesy of the Mayo Clinic Molecular Genetics Laboratory.)

Fig. 5. Fragment-size analysis using CE and fluorescence detection: spinocerebellar ataxia type I. The electopherograms show PCR products using primers flanking the CAG repeat of the ataxin-1 gene. The forward PCR primer is labeled with 6-FAM; the resultant peaks are shown in blue. The PCR products are mixed with a ROX-labeled size marker, shown in red, and coinjected for capillary electrophoresis on an ABI 3100 (Applied Biosystems, Foster City, CA). (A) a patient with 2 normal alleles, 30 and 36 repeats (222 and 239 bases, respectively; (B) a patient with normal allele of 26 repeats (211 bases) and 1 expanded allele of 68 repeats (332 bases). This expansion is diagnostic of spinocerebellar ataxia type I. (Courtesy of the Mayo Clinic Molecular Genetics Laboratory.)

Sscp Satellite
Fig. 6. The number of PubMed citations for SSCP by year since the original description in 1989.

of the gene to be studied, denaturation of the double-stranded PCR product by heat, and electrophoresis on a nondenaturing polyacrylamide gel (15). During the electrophoresis, the single-stranded DNA fragments fold into a three-dimensional shape according to their primary sequence. The separation then becomes a function of the shape of the single-stranded molecules. If wild-type and mutant PCR products differ in their sequence, even by only a single nucleotide, they will likely adopt different three-dimensional structures and exhibit different electrophoretic mobilities (Fig. 7). Figure 8 shows SSCP analysis of exon 8 of the p53 gene from three breast tumor cell lines (16,17).

In order to prevent the two single strands from reannealing to form double-stranded DNA, the concentration of DNA in the loading buffer is kept very low. Thus, in order to visualize the bands, radioactive labeling of the DNA is typically required. However, as an alternative, silver staining seems to give adequate sensitivity and is increasing in popularity.

The issue of sensitivity of mutation scanning methods (i.e., the percentage of mutations detected) is a difficult one. Sensitivity is likely to be influenced by many factors, including, but not limited to; type of base substitution, length of the fragment examined, the local base sequences, the G+C content of the DNA fragment, and the location of the sequence variation relative to the ends of the fragment. For each of the mutation scanning methods, with the sole exception of denaturing gradient gel electrophoresis, there is no precise theory that can be used to predict whether a given method will detect a particular mutation. Thus, the only determinations of sensitivity of mutation scanning methods have been empirical. Typically, studies of sensitivity have used a set of previously characterized mutations found in the gene that the author is studying. Few of the above-listed variables are addressed in most studies, and no study to date has addressed them all.

The reported sensitivity of SSCP has ranged from 35% to near 100%. Sheffield and colleagues used a set of artificial mutants originally created by Myers et al. to study the effect of sequence variation on the promoter region of the mouse P-globin gene (18,19). They demonstrated a pronounced effect of fragment length on sensitivity. At low fragment sizes (100-150 bp), SSCP had a sensitivity approaching 100%; however, when the length increased to 500 bp, the sensitivity dropped to approx 50%.

More recent studies by Highsmith and colleagues have attempted to examine a number of the parameters affecting the sensitivity of SSCP. In order to address all of these issues, this group prepared a set of plasmid constructs with inserts, derived from common cloning vectors, with G+C contents of 40%, 50%, and 60%. Then, using site-directed mutatgenesis, all four bases were introduced into the same position in each set of constructs. With a battery of PCR primers, the investigators could examine the effects of G+C content, fragment size, specific base change, and position of the mutation relative to the end of the fragment on sensitivity of SSCP. Using this "DNA Toolbox," this group found that the primary determinants of sensitivity were the G+C content of the PCR fragment and the temperature of the gel during electrophoresis (20).

Running the SSCP gels under different conditions has been reported to increase sensitivity, as has running the gels on

Fig. 7. Schematic representation of single-stranded conformational polymorphism analysis. In SSCP analysis, the electrophoretic mobilities of the single-stranded DNA species are a function of their three-dimensional conformation. This conformation is determined by the most thermo-dynamically favored intrastrand base-pairing. This, in turn, is directly determined by the primary sequence. Thus, if two DNA fragments differ in sequence, they will fold into different conformations and exhibit different electrophoretic mobilities.

Fig. 7. Schematic representation of single-stranded conformational polymorphism analysis. In SSCP analysis, the electrophoretic mobilities of the single-stranded DNA species are a function of their three-dimensional conformation. This conformation is determined by the most thermo-dynamically favored intrastrand base-pairing. This, in turn, is directly determined by the primary sequence. Thus, if two DNA fragments differ in sequence, they will fold into different conformations and exhibit different electrophoretic mobilities.

Pregnancy Guide

Pregnancy Guide

A Beginner's Guide to Healthy Pregnancy. If you suspect, or know, that you are pregnant, we ho pe you have already visited your doctor. Presuming that you have confirmed your suspicions and that this is your first child, or that you wish to take better care of yourself d uring pregnancy than you did during your other pregnancies; you have come to the right place.

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