I Tiemann-Boege, C Curtis, DN Shinde, DB Goodman, S Tavaré, N Arnheim
The amplification of millions of single molecules in parallel can be performed on microscopic magnetic beads that are contained in aqueous compartments of an oil-buffer emulsion. These bead-emulsion amplification (BEA) reactions result in beads that are covered by almost-identical copies derived from a single template. The post-amplification analysis is performed using different fluorophore-labeled probes. We have identified BEA reaction conditions that efficiently produce longer amplicons of up to 450 base pairs. These conditions include the use of a Titanium Taq amplification system. Second, we explored alternate fluorophores coupled to probes for post-PCR DNA analysis. We demonstrate that four different Alexa fluorophores can be used simultaneously with extremely low crosstalk. Finally, we developed an allele-specific extension chemistry that is based on Alexa dyes to query individual nucleotides of the amplified material that is both highly efficient and specific.