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 into aqueous microreactors in a water-in-oil emulsion such that a plurality of aqueous microreactors comprise a single copy of a nucleic acid, a single bead capable of binding to the nucleic acid, and amplification reaction solution containing reagents necessary to perform nucleic acid amplification;
(b) amplifying the nucleic acids in the microreactors to form amplified copies of said nucleic acids and binding the amplified copies to beads in the microreactors;
(c) delivering the beads to areas on a planar surface to produce an array of at least 10,000 reaction sites; and
(d) performing a sequencing reaction simultaneously on the nucleic acids at the sites.
2. The method of claim 1 wherein the reaction sites have a center to center spacing of 20 to 100 μm.
3. The method of claim 1 wherein the nucleic acids are 30-500 bases.
4. The method of claim 1 wherein a plurality of the beads bind at least 10,000 amplified copies.
5. The method of claim 1 wherein step (b) is accomplished using polymerase chain reaction.
6. The method of claim 1 wherein the sequencing reaction is a pyrophosphate-based sequencing reaction.
7. The method of claim 1 wherein the sequencing reaction comprises the steps of:
(i) annealing an effective amount of a sequencing primer to the amplified copies of the nucleic acid 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 a 3′ end of said sequencing primer, a sequencing reaction byproduct; and
(ii) identifying the sequencing reaction byproduct, thereby determining the sequence of the nucleic acid in a plurality of the reaction chambers.
8. The method of claim 1 further comprising the steps of:
(i) uniquely tagging nucleic acids from different biological sources to create libraries of nucleic acids with different detectable sequence tags;
(ii) sequencing said nucleic acids and detecting said detectable sequence tag from each said tagged nucleic acid.
9. The method of claim 8 wherein the libraries are delivered to the aqueous microreactors individually or wherein the libraries are mixed and delivered to the aqueous microreactors simultaneously.
10. The method of claim 8 wherein said detectable sequence tag comprises an oligonucleotide of between 2 and 50 bases.
11. The method of claim 1 further comprising:
releasing the beads from the microreactors; and
enriching for beads comprising the bound amplified copies.