title: Product length, dye choice, and detection chemistry in the bead-emulsion amplification of millions of single DNA molecules in parallel.
abstract: 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.
authors: Institute of Biophysics, Johannes Kepler University, Linz, 4040 Austria. Irene.Tiemann@jku.at
authors: Irene
authors: I
authors: Tiemann-Boege
authors: Christina
authors: C
authors: Curtis
authors: Deepali N
authors: DN
authors: Shinde
authors: Daniel B
authors: DB
authors: Goodman
authors: Simon
authors: S
authors: Tavaré
authors: Norman
authors: N
authors: Arnheim
mesh_terms: D000042:Absorption; D000483:Alleles; D000818:Animals; D001482:Base Composition; D002417:Cattle; D003116:Color; D004247:DNA / Q000032:analysis* / Q000737:chemistry / Q000235:genetics*; D004655:Emulsions; D005456:Fluorescent Dyes / Q000737:chemistry*; D006801:Humans; D008863:Microspheres; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*
chemical_list: D004655:Emulsions; D005456:Fluorescent Dyes; D004247:DNA