Single-stranded DNA can adopt multiple conformations under non-denaturing conditions. In the absence of a complementary strand, DNA will anneal short internal complementary sequences, forming a complex "knot." A DNA molecule follows a complex path of folding to reach its final form, influenced by the solution environment and temperature. The path consists of a series of annealing steps, each stabilizing and to some extent directing the next. Minor alterations in the sequence of the DNA will disrupt the annealing process and result in a different final shape. The compactness of these structures will determine how fast the single stranded DNA migrates through a non-denaturing gel. Such differences in electrophoretic mobility between nearly identical strands serve as the basis for the technique known as single strand conformation polymorphism (SSCP) analysis, and like heteroduplex analysis, it is a rapid method for mutational screening.
|SSCP (Single Strand Conformational Polymorphism) Analysis. Single point mutations can cause major differences in the folded form of single stranded DNA. These differences can be detected as differences in electrophoretic mobility.|
Samples are denatured with heat and then rapidly cooled. Rapid cooling favors self-annealing, because insufficient time is allowed for complementary strands to collide and orient for duplex formation. The renatured samples are analyzed on a gel opposite the control DNA. As with heteroduplex analysis, all fragments must be the same length. Mutant samples will show a mobility different from the control DNA. The gel matrix used must be optimized for the resolution of DNA conformers of the same length. Various combinations of acrylamide and bis-acrylamide are mentioned in the literature, at ratios from 29 to 1 to 50 to 1, and at percentages from 4 to 8. National Diagnostics' SequaGel MD is optimized to provide superior results in both heteroduplex and SSCP analysis.
SSCP Analysis Procedure
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Но это полный абсурд. Неужели Хейл никогда не слышал о принципе Бергофского. - Вот что нам надо сделать.