Magnetic Bead-Isolated Single-Strand DNA for SSCP Analysis

Single-strand DNA conformational polymorphism (SSCP) makes use of sequencedependent folding and structural conformation assumed by ssDNA for detection of small sequence changes or point mutations in DNA fragments. The technique was developed using polyacrylamide gel electrophoretic (PAGE) fractionation of distinctive conformational isomers (conformers) of ssDNAs. These are formed by denaturation of short polymerase chain reaction (PCR) products (typically <200 bp) (1-7). Conventional understanding of SSCP suggests that conformational distortion created by the folding (loops and bends) stabilized by the formation (short) regions of intramolecular duplex between complementary regions within each single-stranded molecule alters the three dimensional shape of the molecule. Crudely, the migrating DNA strand collides with the sieving polymer media and through the temporary interaction of DNA and sieving media molecules, the mobility of the DNA is reduced (8). Differences in the size or conformation of DNA molecules affect the duration of these sieving interactions. Since the two complementary DNA strands differ in sequence, each strand assumes an independent conformation, which has a characteristic mobility. Thus, minor differences in the sequence between DNAs from two sources, such as point mutations, may affect the conformation assumed by each of the strands and identify the sequence differences as a shift in mobility. High-performance capillary electrophoresis (HPCE) is increasingly used for SSCP analysis, with detection by UV absorption of DNA (9,10), or by LIF of ssDNA tagged with fluorescent dye-labeled primers (11-14).