Methods and apparatus for template capture and normalization for submicroliter reaction
View Patent ↗Methods for preparing nanoscale reactions using nucleic acids are presented. Nucleic acids are captured saturably, yet reversibly, on the internal surface of the reaction chamber, typically a capillary. Excess nucleic acid is removed and the reaction is performed directly within the capillary. Alternatively, the saturably bound nucleic acid is eluted, dispensing a metered amount of nucleic acid for subsequent reaction in a separate chamber. Devices for effecting the methods of the invention and a system designed advantageously to utilize the methods for high throughput nucleic acid sequencing reactions are also provided.
1. A method of performing an enzymatic reaction in a capillary tube using a normalized quantity of DNA, comprising:
introducing an enzymatic reaction mixture into a capillary tube, said tube having a normalized quantity of DNA,
wherein said reaction mixture comprises an oligonucleotide primer, a DNA polymerase, and deoxynucleotide triphosphates (dNTPs),
said DNA having been saturably bound from an excess thereof directly on an inner surface of said capillary tube by contacting said inner surface with a solution comprising DNA and a chaotropic agent for a time sufficient for the DNA to have become saturably bound to said inner surface; and
said excess of DNA having been removed therefrom; and
performing said enzymatic reaction in said capillary tube using said normalized quantity of DNA.
2. The method of claim 1 , further comprising the step of size selecting the DNA prior to said step of introducing said enzymatic reaction mixture into said capillary tube.
3. The method of claim 2 , wherein said step of size-selecting is effected by the exclusion of genomic DNA from the bore of said capillary.
4. The method of claim 1 , wherein said reaction mixture further comprises dideoxynucleotide triphosphates.
5. The method of claim 4 , wherein said dideoxynucleotide triphosphates are conjugated to a fluorophore.
6. The method of claim 5 , wherein said fluorophore is selected from among the group consisting of: fluorescein, 5-carboxy-fluorescein, 6-carboxy-rhodamine, N,N,N′,N′-tetramethyl-5-carboxyrhodamine and 5-carboxy-X-rhodamine, rhodamine 110, rhodamine-6-G, tetramethyl rhodamine and rhodamine X.
7. The method of claim 5 , wherein said fluorophore is an energy transfer fluorophore.
8. The method of claim 1 , wherein said primer is complementary to a plurality of contiguous nucleotides in said DNA.
9. The method of claim 8 , wherein said primer is conjugated to a fluorophore.
10. The method of claim 9 , wherein said fluorophore is selected from among the group consisting of: fluorescein, 5-carboxy-fluorescein, 6-carboxy-rhodamine, N,N,N′,N′-tetramethyl-5-carboxyrhodamine and 5-carboxy-X-rhodamine, rhodamine 110, rhodamine-6-G, tetramethyl rhodamine and rhodamine X.
11. The method of claim 9 , wherein said fluorophore is an energy transfer fluorophore.
12. The method of claim 1 , wherein said DNA polymerase is thermostable.
13. The method of claim 1 , further comprising subjecting said enzymatic reaction mixture to at least one thermal cycle.
14. The method of claim 1 , wherein said DNA is selected from among the group consisting of eukaryotic DNA, prokaryotic DNA, animal DNA, plant DNA, fungal DNA, archea DNA, mammalian DNA, human DNA, virus DNA, bacteriophage DNA, lambda phage DNA, M13 phage DNA, bacterial DNA, E. coli DNA, genomic DNA, plasmid DNA, episomal DNA, DNA produced by polymerase chain reaction and DNA to be spotted onto a substrate to create a microarray.
15. The method of claim 1 , further comprising analyzing a product of said enzymatic reaction to determine the sequence of said DNA.
16. The method of claim 15 , wherein a product of said enzymatic reaction is purified prior to said step of analyzing said product to determine the sequence of said DNA.
17. The method of claim 16 , wherein said purification is effected using a technique selected from among the group consisting of alcohol precipitation, size exclusion chromatography, size exclusion chromatography using Sephadex® and treatment with combination of enzymes.
18. The method of claim 16 , wherein said step of analyzing a product of said enzymatic reaction to determine the sequence of said DNA is effected using a technique selected from among the group consisting of electrophoresis, capillary electrophoresis, gel electrophoresis, mass spectroscopy, MALDI mass spectroscopy, SELDI mass spectroscopy, fluorescence detection, scanning confocal laser-induced fluorescence detection and analytical microchip analysis.
19. The method of claim 16 , wherein said step of analyzing a product of said enzymatic reaction to determine the sequence of said DNA is effected by single base extension reactions.