| 主权项 |
1. A method for amplifying a region of analyte DNA molecules, comprising:
amplifying a region of analyte DNA molecules using a high fidelity DNA polymerase to form a set of first amplicons; forming microemulsions comprising said first amplicons and reagent beads, wherein the reagent beads are bound to a plurality of molecules of a primer for amplifying the set of first amplicons; amplifying the first amplicons in the microemulsions, whereby product beads are formed which are bound to a plurality of copies of second amplicons; breaking the microemulsions; amplifying the second amplicons bound to the product beads using rolling circle amplification to form third amplicons, wherein the rolling circle amplification employs a circularizable probe, said probe comprising a first and a second region of complementarity with a third and a fourth region on said second amplicons, wherein said first and second regions are non-contiguous on said probe, and wherein said third and fourth regions are non-contiguous on said second amplicons, wherein said second amplicons comprise a fifth region of 1-30 nucleotides between said third and fourth regions, wherein upon hybridization of the circularizable probe to the second amplicons a DNA polymerase fills in 1-30 nucleotides complementary to the fifth region, between said first and second region of complementarity of the circularizable probe, and template-driven ligation of the ends of the circularizable probe forms a circle; and analyzing single beads to the exclusion of bead doublets and bead aggregates. |
