What is MSSCP?
MSSCP (Multitemperature Single Strand Conformation Polymorphism) is a novel method designed for detection of minor genetic variants underlying many diseases and conditions. The method was proven to disclose variants present in highly heterogeneous tumor tissue at the level of 1%.
Multitemperature Single Stand Conformation Polymorphism (MSSCP) is a simple and reliable technique that can be used for detection of point mutations and larger rearrangements in nucleic acids. The technique works well for analysis of the PCR fragments of 100-350 bp in length.
Figure 1. The MSSCP analysis of the PCR products covering the exon 1 of KCN1 gene. Wild Type, G226A/WT heterozygotes and G226A homozygotes are visible.
The MSSCP technology relies on the physical property of nucleic acids: single-stranded nucleic acid acquires a characteristic 3-dimensional structure in native conditions depending on its nucleotide sequence. Such structures can be next distinguished by their different electrophoretic mobility resulting from genetic alterations. The MSSCP is a great improvement of SSCP (single strand conformation polymorphism) developed by Orita et al. in 1989, widely used technique for mutation detection, however one of the main drawbacks is relatively low reproducibility and poor sensitivity as well. Single-stranded nucleic acids that differ by one or more nucleotides can sometimes possess that same electrophoretic mobility that is in turn responsible for the mutation detection rate typically ranging from 70 to 90% (Murakami et al., 1991; Sheffield et al., 1991; Hongyo et al., 1993). Main physical conditions influencing ssDNA conformers and SSCP patterns are: pH, ionic strength and temperature.
In Multitemperature SSCP (MSSCP), an electrophoretic separation is performed under sequentially changed gel temperature. This results in a significantly higher mutation detection rate in reduced time of analysis. There is no need to repeat electrophoresis in various temperatures as in classic SSCP. Temperature changes increase the probability for the PCR products to adopt different ssDNA conformations during the electrophoretic run if they carry any mutation and thus increase the mutation detection rate.
All kinds of genetic alterations can be detected using the MSSCP technology. No prior need for knowledge of the mutation type is required. This makes MSSCP an extremely useful tool, whenever a large number of samples are screened for known and unknown mutations in single loci.