Closed-loop biochemical analyzers

A method of sequencing a target nucleic acid, comprising:

• (a) providing an integrated microfluidic system comprising a microfluidic device comprising:
• at least a first sequencing reaction channel and at least a first sequencing reagent introduction channel, the sequencing reaction channel and sequencing reagent introduction channel being in fluid communication;
• and,
• a fluidic interface in fluid communication with the sequencing reagent introduction channel for sampling a plurality of sequencing reagents or mixtures of sequencing reagents from a plurality of sources of sequencing reagents or mixtures of sequencing reagents and introducing the sequencing reagents or mixtures of sequencing reagents into the sequence reagent introduction channel from the sources of sequencing reagents or mixtures of sequencing reagents;
• selecting a first sequencing primer sequence complementary to a first subsequence of a first target nucleic acid sequence;
• (b) introducing the first sequencing primer and the first target nucleic acid sequence into the sequence reagent introduction channel;
• (c) hybridizing the first primer sequence to the first subsequence in the first sequencing reaction channel and polymerase-extending the first primer sequence along the length of the target nucleic acid sequence to form a first extension product that is complementary to the first subsequence and a second subsequence of the target nucleic acid;
• (d) determining the sequence of the first extension product;
• (e) based upon the sequence of the first extension product, selecting a second primer sequence complementary to the second subsequence of the target nucleic acid sequence;
• (f) hybridizing the second primer sequence to the second subsequence in the first sequencing reaction channel, or, optionally, hybridizing the second primer sequence in the second subsequence in a second sequencing reaction channel;
• (g) extending the second primer sequence along the length of the target nucleic acid sequence to form a second extension product that is complementary to the second subsequence and a third subsequence of the target nucleic acid sequence;
• and
• (h) determining the sequence of the second extension product.

A method of sequencing a nucleic acid comprising:

• providing a set of sequencing primers, a target nucleic acid, a polymerase, dNTPs, and ddNTPs;
• selecting a first primer from the set of primers;
• introducing the first primer into a microfluidic device;
• mixing the first primer, the polymerase, the dNTPs, and the ddNTPs in a first zone of the microfluidic device under conditions permitting target dependent polymerization of the dNTPs, thereby providing polymerization products;
• separating polymerization products by size in a second zone of the microfluidic device to provide at least a first portion of the sequence of the target nucleic acid;
• selecting a second primer from the set of primers, which primer is complementary to the first portion of the target nucleic acid;
• introducing the second primer into the microfluidic device;
• mixing the second primer, the polymerase, the dNTPs, and the ddNTPs in a third zone of the microfluidic device under conditions permitting target dependent polymerization of the dNTPs, thereby providing polymerization products;
• separating polymerization products by size in a fourth zone of the microfluidic device to provide at least a second portion of the sequence of the target nucleic acid.

An integrated method of performing a fluidic analysis of sample materials, comprising:

• (a) providing an integrated microfluidic system comprising a microfluidic device comprising:
• at least a first reaction chamber or channel, and at least a first reagent introduction channel, the first reaction chamber or channel and reagent introduction channel being in fluid communication;
• a material transport system for controllably transporting a material through the reagent introduction channel and reaction chamber or channel;
• a fluidic interface in fluid communication with the reagent introduction channel for sampling a plurality of reagents or mixtures of reagents from a plurality of sources of reagents or mixtures of reagents and introducing the reagents or mixtures of reagents into the reagent introduction channel;
• (b) selecting a first set of DNA sequencing reagents comprising a first nucleic acid sequencing primer from the plurality of sources of sequencing reagents or mixtures of sequencing reagents;
• (c) introducing a first DNA template and the first set of DNA sequencing reagents comprising a first nucleic acid sequencing primer into the first reaction chamber or channel whereupon the first DNA template and the first set of DNA sequencing reagents comprising a first nucleic acid sequencing primer react to produce a first set of DNA sequencing products;
• (d) analyzing the first set of DNA sequencing products by separating the DNA sequencing products by size and detecting the size separated DNA sequencing products, thereby providing sequence information for the first DNA template;
• (e) based upon the the sequence information determined in step (d), selecting a second set of DNA sequencing reagents comprising a second sequencing primer and a second DNA template;
• (f) introducing the second set of DNA sequencing reagents comprising a second sequencing primer and the first DNA template into the first reaction chamber or channel, or, optionally, into a second reaction chamber or channel in the microfluidic device, whereupon the second set of DNA sequencing reagents comprising a second sequencing primer and the first DNA template react to produce a second set of DNA sequencing products;
• and
• (g) analyzing the second set of DNA sequencing products to provide additional sequence information for the first DNA template.

A method of performing iterative fluid operations, comprising:

• providing a microfluidic system which comprises:
• a microfluidic device having at least a first microscale channel disposed therein, the at least first channel being in fluid communication with a plurality of reagent sources;
• a material transport system for transporting reagents from each of the plurality of reagent sources into the at least first channel;
• introducing at least a first nucleic acid template from at least a first reagent source and a first sequencing primer from at least a second reagent source into the at least first channel, the nucleic acid template and first sequencing primer producing a first elongation product;
• detecting a nucleotide sequence of the first elongation product;
• and
• based upon the reaction of the first and second reagents, selecting and introducing a an additional amount of the nucleic acid template and a second sequencing primer into the at least first channel the second sequencing primer based upon the nucleotide sequence of the first elongation product.