Multiplex, fluorescent single-strand conformation polymorphism using stepped polymerase chain reaction primers.

A method for high-throughput screening for a well-characterized mutation at the APC locus was developed using stepped PCR primers, pooled product, and fluorescent single-strand conformation polymorphism (SSCP). Using a panel of two known normals, three known heterozygous mutants, and one known homozygous mutant, eight stepped PCR products were developed that showed the same mobility shift on a PE Biosystems 373A. Using this method, up to eight different samples can be pooled for same lane analysis.

Considerations in adding mutation detection services to a sequencing core facility.

Because analysis of single nucleotide polymorphisms (SNPs) can be invaluable in understanding genomic variation and the genetic basis of disease, there is a need for high-throughput, high-accuracy mutation detection methods for identifying SNPs. A sequencing core facility can enhance the services it offers by providing genome analysis methods to search for informative SNPs. Denaturing high-performance liquid chromatography and single-strand conformation polymorphism analysis are methods of mutation detection that are amenable to a sequencing core environment. They are useful for screening large sample sets to identify novel SNPs, eliminating the need to sequence every sample in the set. These methods allow analysis of more samples than would otherwise be economically feasible by sequencing alone.