DIGITAL PCR CALIBRATION FOR HIGH THROUGHPUT SEQUENCING

摘要

Disclosed is a method for accurately determining the number of template molecules in a library of nucleic acids (e.g., DNA) to be sequenced. The method does not require large amounts of the DNA sample, nor does it require the preparation of a standard curve. The method is especially applicable to methodologies for "sequencing by synthesis," where quantitation of the starting library is important. The method uses quantitative real time PCR, especially digital PCR, which measures the number of individual molecules in a sample. The present method particularly may use a microfluidic device for running large numbers of PCR reactions. Each PCR reaction is monitored in real time by a primer/probe combination. The forward primer is adapted to contain a sequence not on the adapter but which corresponds to a probe sequence. A short probe which generates fluorescence during the PCR process is used.