Methods of amplifying and sequencing nucleic acids
An apparatus and method for performing rapid DNA sequencing, such as genomic sequencing, is provided herein. The method includes the steps of preparing a sample DNA for genomic sequencing, amplifying the prepared DNA in a representative manner, and performing multiple sequencing reaction on the amplified DNA with only one primer hybridization step.
1. A method for sequencing nucleic acids comprising:
(a) delivering the nucleic acids to an array of at least 10,000 reaction chambers on a planar -surface, such that a plurality of the reaction chambers comprise beads capable of binding the nucleic acid and no more than one nucleic acid;
(b) amplifying the nucleic acids in the reaction chambers, wherein a plurality of amplified copies of the nucleic acids are immobilized to beads; and
(c) performing a sequencing reaction simultaneously in a plurality of the reaction chambers comprising the immobilized amplified copies of the nucleic acids.
2. The method of claim 1 wherein the reaction chambers have a center to center spacing of between 20 to 100 μm.
3. The method of claim 1 wherein the nucleic acids are between 30-500 bases.
4. The method of claim 1 wherein the amplifying step is accomplished using polymerase chain reaction.
5. The method of claim 1 wherein the sequencing reaction is a pyrophosphate-based sequencing reaction.
6. The method of claim 1 wherein the sequencing reaction comprises the steps of:
(a) annealing an effective amount of a sequencing primer to immobilized amplified copies of the nucleic acids and extending the sequencing primer with a polymerase and a predetermined nucleotide triphosphate to yield a sequencing product and, if the predetermined nucleotide triphosphate is incorporated onto the 3′ end of said sequencing primer, a sequencing reaction byproduct; and
(b) identifying the sequencing reaction byproduct, thereby determining the sequence of the nucleic acid in a plurality of the reaction chambers.
7. The method of claim 1 wherein the reaction chambers are cavities formed by etching one end of a fiber optic bundle.