Microsystem for rapid DNA sequencing
First Claim
1. A method for sequencing DNA, comprising the sequential steps of:
- (a) covalently linking the DNA to a first compound;
(b) transferring the DNA into a capillary having a volume less than about 1 μ
L, wherein a second compound is bound to the inner wall of the capillary, wherein the first compound binds to the second compound with high affinity, whereby the DNA becomes immobilized on the inner wall of the capillary;
(c) synthesizing oligonucleotides complementary to all or a portion of the immobilized DNA with a DNA polymerase and DNA primers;
wherein the oligonucleotides are substantially identical to one another, except that the 3'"'"' ends of the oligonucleotides are a variable distance from a common 5'"'"' end, so that the oligonucleotides have variable lengths;
wherein the terminal nucleotide base on the 3'"'"' end of the oligonucleotides is linked to one of four labels that is spectroscopically detectable, and that identifies the nucleotide base to which it is linked; and
wherein the oligonucleotides are synthesized within the capillary containing the immobilized DNA;
(d) transferring the oligonucleotides to a microfabricated capillary electrophoresis column, and electrophoretically separating the oligonucleotides within the column by size; and
(e) spectroscopically detecting and identifying the labels linked to the electrophoretically separated oligonucleotides;
whereby the order in which the labels of the electrophoresed oligonucleotides are detected corresponds to the sequence of at least a portion of the DNA.
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Abstract
A system is disclosed for the rapid and cost-effective sequencing of DNA. There are three principal components of the system: (1) a microreactor, which prepares DNA sequencing "ladders" using solid-phase techniques, preferably in capillary tubes whose volumes are on the order of 10-1000 nanoliters, preferably 10-200 nanoliters; (2) a microfabricated electrophoresis capillary separation unit; and (3) a fluorescence detector with single-mode optical fibers interfaced directly to the electrophoresis capillary. The system is suitable for a highly multiplexed, automated DNA sequencing device Typical. steps in sequencing are as follows: (1) PCR amplification of a DNA template in microtiter dishes using labelled primers, e.g., primers labelled with biotin; (2) immobilizing the labelled PCR products on the walls of one or more capillary tubes having volumes on the order of 10-200 nanoliters; (3) preparing nanoliter quantities of labelled Sanger extension products of the amplified DNA; (4) purifying the oligonucleotide sequencing ladders; (5) high speed electrophoretic separation of the sequencing ladders; and (6) near-infrared, laser-induced fluorescence detection of the oligonucleotides. Base-calling is preferably performed in a single lane format with a single fluorophore, in which the bases are distinguished by different fluorescence lifetimes of dyes that otherwise have similar absorption and fluorescence emission spectra at the wavelengths used. Typical read lengths are on the order of 400-500 bases. Fluorescence is performed on-chip with one single-mode optical fiber carrying the excitation light to the capillary channel, and a second single-mode optical fiber collecting the fluorescent photons. Only sub-microliter volumes of expensive sequencing reagents and dye-labeled ddNTPs are required in this system.
302 Citations
39 Claims
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1. A method for sequencing DNA, comprising the sequential steps of:
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(a) covalently linking the DNA to a first compound; (b) transferring the DNA into a capillary having a volume less than about 1 μ
L, wherein a second compound is bound to the inner wall of the capillary, wherein the first compound binds to the second compound with high affinity, whereby the DNA becomes immobilized on the inner wall of the capillary;(c) synthesizing oligonucleotides complementary to all or a portion of the immobilized DNA with a DNA polymerase and DNA primers;
wherein the oligonucleotides are substantially identical to one another, except that the 3'"'"' ends of the oligonucleotides are a variable distance from a common 5'"'"' end, so that the oligonucleotides have variable lengths;
wherein the terminal nucleotide base on the 3'"'"' end of the oligonucleotides is linked to one of four labels that is spectroscopically detectable, and that identifies the nucleotide base to which it is linked; and
wherein the oligonucleotides are synthesized within the capillary containing the immobilized DNA;(d) transferring the oligonucleotides to a microfabricated capillary electrophoresis column, and electrophoretically separating the oligonucleotides within the column by size; and (e) spectroscopically detecting and identifying the labels linked to the electrophoretically separated oligonucleotides; whereby the order in which the labels of the electrophoresed oligonucleotides are detected corresponds to the sequence of at least a portion of the DNA. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19)
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20. An apparatus for sequencing DNA, using first and second compounds that bind to one another with high affinity, said apparatus comprising:
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(a) a capillary having a volume less than about 200 nanoliters, wherein the inner wall of said capillary binds or is capable of binding the second compound, and wherein said capillary is adapted to immobilize DNA that is linked to the first compound by allowing the first and second compounds to bind to one another, whereby the DNA becomes immobilized on the inner wall of said capillary;
wherein said capillary is capable of acting as a reaction vessel for synthesizing, within said capillary, oligonucleotides complementary to all or a portion of the immobilized DNA with a DNA polymerase and DNA primers;
wherein the oligonucleotides are substantially identical to one another, except that the 3'"'"' ends of the oligonucleotides are a variable distance from a common 5'"'"' end, so that the oligonucleotides have variable lengths; and
wherein the terminal nucleotide base on the 3'"'"' end of the oligonucleotides is linked to a label that is spectroscopically detectable, and that identifies the nucleotide base to which it is linked;(b) a microfabricated capillary electrophoresis column connected to said capillary to receive the synthesized oligonucleotides, and to electrophoretically separate the oligonucleotides within the column by size; and (c) a detector to spectroscopically detect and identify the labels linked to the electrophoretically separated oligonucleotides; whereby the order in which the labels of the electrophoresed oligonucleotides are detected corresponds to the sequence of at least a portion of the DNA.
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21. An apparatus for sequencing DNA, using first and second compounds that bind to one another with high affinity, said apparatus comprising:
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(a) a capillary having a volume less than about 1 μ
L, wherein the inner wall of said capillary binds or is capable of binding the second compound, and wherein said capillary is adapted to immobilize DNA that is linked to the first compound by allowing the first and second compounds to bind to one another, whereby the DNA becomes immobilized on the inner wall of said capillary;
wherein said capillary is capable of acting as a reaction vessel for synthesizing, within said capillary, oligonucleotides complementary to all or a portion of the immobilized DNA with a DNA polymerase and DNA primers;
wherein the oligonucleotides are substantially identical to one another, except that the 3'"'"' ends of the oligonucleotides are a variable distance from a common 5'"'"' end, so that the oligonucleotides have variable lengths; and
wherein the terminal nucleotide base on the 3'"'"' end of the oligonucleotides is linked to a label that is spectroscopically detectable, and that identifies the nucleotide base to which it is linked;(b) a microfabricated capillary electrophoresis column connected to said capillary to receive the synthesized oligonucleotides, and to electrophoretically separate the oligonucleotides within the column by size; and (c) a detector to spectroscopically detect and identify the labels linked to the electrophoretically separated oligonucleotides;
wherein said detector comprises a first single-mode optic fiber to transmit near-infrared laser light through the electrophoretically separated oligonucleotides to excite the labels of the electrophoretically separated oligonucleotides; and
a second single-mode optic fiber to collect near-infrared fluorescent photons from the labels on the electrophoretically separated oligonucleotides;
wherein the two said optic fibers are positioned at approximately 90°
relative to one another; and
wherein each of the two said optic fibers is positioned at approximately 90°
relative to the direction of electrophoretic travel of the oligonucleotides;whereby the order in which the labels of the electrophoresed oligonucleotides are detected corresponds to the sequence of at least a portion of the DNA.
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22. An apparatus for sequencing DNA, using first and second compounds that bind to one another with high affinity, said apparatus comprising:
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(a) a capillary having a volume less than about 1 μ
L, wherein the inner wall of said capillary binds or is capable of binding the second compound, and wherein said capillary is adapted to immobilize DNA that is linked to the first compound by allowing the first and second compounds to bind to one another, whereby the DNA becomes immobilized on the inner wall of said capillary;
wherein said capillary is capable of acting as a reaction vessel for synthesizing, within said capillary, oligonucleotides complementary to all or a portion of the immobilized DNA with a DNA polymerase and DNA primers;
wherein the oligonucleotides are substantially identical to one another, except that the 3'"'"' ends of the oligonucleotides are a variable distance from a common 5'"'"' end, so that the oligonucleotides have variable lengths; and
wherein the terminal nucleotide base on the 3'"'"' end of the oligonucleotides is linked to a label that is spectroscopically detectable, and that identifies the nucleotide base to which it is linked;(b) a microfabricated capillary electrophoresis column connected to said capillary to receive the synthesized oligonucleotides, and to electrophoretically separate the oligonucleotides within the column by size, wherein said capillary electrophoresis column is fabricated in an x-ray resist substrate, wherein the x-ray resist has no substantial surface charge, and wherein the x-ray resist comprises a polyacrylate; and (c) a detector to spectroscopically detect and identify the labels linked to the electrophoretically separated oligonucleotides; whereby the order in which the labels of the electrophoresed oligonucleotides are detected corresponds to the sequence of at least a portion of the DNA. - View Dependent Claims (23)
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24. An apparatus for sequencing DNA, using first and second compounds that bind to one another with high affinity, said apparatus comprising:
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(a) a capillary having a volume less than about 1 μ
L, wherein the inner wall of said capillary binds or is capable of binding the second compound, and wherein said capillary is adapted to immobilize DNA that is linked to the first compound by allowing the first and second compounds to bind to one another, whereby the DNA becomes immobilized on the inner wall of said capillary;
wherein said capillary is capable of acting as a reaction vessel for synthesizing, within said capillary, oligonucleotides complementary to all or a portion of the immobilized DNA with a DNA polymerase and DNA primers;
wherein the oligonucleotides are substantially identical to one another, except that the 3'"'"' ends of the oligonucleotides are a variable distance from a common 5'"'"' end, so that the oligonucleotides have variable lengths; and
wherein the terminal nucleotide base on the 3'"'"' end of the oligonucleotides is linked to a label that is spectroscopically detectable, and that identifies the nucleotide base to which it is linked;(b) a microfabricated capillary electrophoresis column connected to said capillary to receive the synthesized oligonucleotides, and to electrophoretically separate the oligonucleotides within the column by size, wherein said capillary electrophoresis column is fabricated in an x-ray resist substrate, wherein the x-ray resist has no substantial surface charge, and wherein the x-ray resist comprises a poly(diene sulfone); and (c) a detector to spectroscopically detect and identify the labels linked to the electrophoretically separated oligonucleotides; whereby the order in which the labels of the electrophoresed oligonucleotides are detected corresponds to the sequence of at least a portion of the DNA. - View Dependent Claims (25)
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26. An apparatus for sequencing DNA, using first and second compounds that bind to one another with high affinity, said apparatus comprising:
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(a) a capillary having a volume less than about 1 μ
L, wherein the inner wall of said capillary binds or is capable of binding the second compound, and wherein said capillary is adapted to immobilize DNA that is linked to the first compound by allowing the first and second compounds to bind to one another, whereby the DNA becomes immobilized on the inner wall of said capillary;
wherein said capillary is capable of acting as a reaction vessel for synthesizing, within said capillary, oligonucleotides complementary to all or a portion of the immobilized DNA with a DNA polymerase and DNA primers;
wherein the oligonucleotides are substantially identical to one another, except that the 3'"'"' ends of the oligonucleotides are a variable distance from a common 5'"'"' end, so that the oligonucleotides have variable lengths; and
wherein the terminal nucleotide base on the 3'"'"' end of the oligonucleotides is linked to a label that is spectroscopically detectable, and that identifies the nucleotide base to which it is linked;(b) a microfabricated capillary electrophoresis column connected to said capillary to receive the synthesized oligonucleotides, and to electrophoretically separate the oligonucleotides within the column by size, wherein said capillary electrophoresis column is fabricated in an x-ray resist substrate, wherein the x-ray resist has no substantial surface charge, and wherein the x-ray resist comprises poly(butene-1-sulfone); and (c) a detector to spectroscopically detect and identify the labels linked to the electrophoretically separated oligonucleotides; whereby the order in which the labels of the electrophoresed oligonucleotides are detected corresponds to the sequence of at least a portion of the DNA.
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27. An apparatus for sequencing DNA, using first and second compounds that bind to one another with high affinity, said apparatus comprising:
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(a) a capillary having a volume less than about 1 μ
L, wherein the inner wall of said capillary binds or is capable of binding the second compound, and wherein said capillary is adapted to immobilize DNA that is linked to the first compound by allowing the first and second compounds to bind to one another, whereby the DNA becomes immobilized on the inner wall of said capillary;
wherein said capillary is capable of acting as a reaction vessel for synthesizing, within said capillary, oligonucleotides complementary to all or a portion of the immobilized DNA with a DNA polymerase and DNA primers;
wherein the oligonucleotides are substantially identical to one another, except that the 3'"'"' ends of the oligonucleotides are a variable distance from a common 5'"'"' end, so that the oligonucleotides have variable lengths; and
wherein the terminal nucleotide base on the 3'"'"' end of the oligonucleotides is linked to a label that is spectroscopically detectable, and that identifies the nucleotide base to which it is linked;(b) a microfabricated capillary electrophoresis column connected to said capillary to receive the synthesized oligonucleotides, and to electrophoretically separate the oligonucleotides within the column by size, wherein said capillary electrophoresis column is fabricated in an x-ray resist substrate, wherein the x-ray resist has no substantial surface charge, and wherein the x-ray resist comprises an epoxy resist; and (c) a detector to spectroscopically detect and identify the labels linked to the electrophoretically separated oligonucleotides; whereby the order in which the labels of the electrophoresed oligonucleotides are detected corresponds to the sequence of at least a portion of the DNA. - View Dependent Claims (28)
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- 29. A capillary electrophoresis column microfabricated in an x-ray resist substrate, wherein the x-ray resist has no substantial surface charge, and wherein the x-ray resist comprises a polyacrylate.
- 31. A capillary electrophoresis column microfabricated in an x-ray resist substrate, wherein the x-ray resist has no substantial surface charge, and wherein the x-ray resist comprises a poly(diene sulfone).
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33. A capillary electrophoresis column microfabricated in an x-ray resist substrate, wherein the x-ray resist has no substantial surface charge, and wherein the x-ray resist comprises poly(butene-1-sulfone).
- 34. A capillary electrophoresis column microfabricated in an x-ray resist substrate, wherein the x-ray resist has no substantial surface charge, and wherein the x-ray resist comprises an epoxy resist.
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36. A capillary micro-electrophoresis column comprising an integrated optical system for detecting a fluorescent component of a sample in said column, said integrated optical system comprising a first single mode optic fiber for delivering laser excitation light to the sample in said column, and a second single mode optic fiber for collecting fluorescent photons from the sample in said column and delivering the fluorescent photons to a detector, wherein said first and second optic fibers are positioned at approximately 90°
- relative to one another.
- View Dependent Claims (37, 38, 39)
Specification