By R. G. H. Cotton (Editor), E. Edkins (Editor), S. Forrest (Editor)
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Additional info for Mutation Detection: A Practical Approach (Practical Approach Series)
Sample text
Met, heteroduplex. 2: Single-stranded conformation polymorphism in either the ss or ds regions. Lanes 1, 5-7, 13-15 contain samples that are wild-type in this region. Figure 1B displays analysis of samples that are heterozygous (lanes 1, 13-15) or homozygous (lane 7) for a 32 bp deletion in CCR5. The deletion can be visualized as a higher mobility fragment in the ds region. Note that the heteroduplexes (present only in the heterozygotes) are greatly retarded and nearly overlap the ss region. SSCP alterations are also observed.
7. Applications Since the invention of the PCR-SSCP technique in 1989, many applications of the method have been demonstrated. SSCP has been used to identify alterations in tumour DNA samples as well as mutations in human disease genes and their animal counterparts (1, 8-11). SSCP can be applied to RNA or to reverse transcribed RNA (cDNA) either to increase sensitivity or to assay multiple exons simultaneously (12, 13). SSCP has been applied to complex loci with many alleles, such as the HLA locus (14).
The variables that have been employed are as follows: (a) Temperature: i. room temperature overnight at 15 W constant power for a total of 7000-9000 volt hours ii. 50 W 3-5 h in a 4°C cold room (b) Cross-linking: i 19:1 (5% C) ii. 6% C) iii. 3% C) iv. 5 X MDE (Avitech Diagnostics) (c) Additives: i. 5-10% glycerol ii. 10% sucrose Each of these variables can alter the conformation of single-stranded molecules. SSCP was initially run on 5% acrylamide gels with 5% bisacrylamide cross-linker (5% C) at either room temperature or 4°C with or without glycerol.