Apparatus and methods for sequencing nucleic acids in microfluidic systems

Abstract

The present invention is generally directed to microfluidic systems and methods of using such systems in the determination of the nucleotide sequence of target nucleic acid sequences (referred to herein as the “target”). In particular, the present invention provides methods and systems for determining the relative positions within a target nucleic acid sequence that are occupied by a given nucleotide, e.g., A, T, G or C, by separating mixtures of nested sets of fragments of the target nucleic acid, which sets each include fragments that terminate in a different given nucleotide.

What is claimed is:  
     
         1 . A microfluidic system for determining relative positions in a target nucleic acid sequence that are occupied by a given nucleotide, comprising: 
 a microscale separation channel having first and second ends for separating nucleic acid fragments by size;    a first source of a first nested set of fragments of the target nucleic acid sequence in fluid communication with the separation channel, each of the fragments in the first nested set terminating at a different position occupied by the given nucleotide;    a second source of a second nested set of fragments of the target nucleic acid sequence in fluid communication with the separation channel, each of the fragments in the second nested set terminating in a nucleotide different from the given nucleotide;    a means for mixing a first concentration of the first nested set with a second concentration of the second nested set in a first mixture, the first concentration being determinably different from the second concentration;    a means for injecting a portion of the first mixture into the separation channel and transporting the portion through the separation channel; and    a means for detecting separate fragments of the first and second nested sets in the separation channel.