Heterologous DNA barcoding method

Patent metadata

Field	Value
Publication number	US-9850482-B2
Application number	US-201414772914-A
Country	US
Kind code	B2
Filing date	Mar 7, 2014
Priority date	Mar 7, 2013
Publication date	Dec 26, 2017
Grant date	Dec 26, 2017

Abstract

A heterologous DNA barcoding method is provided. The method includes (a) providing a DNA microarray having DNA oligonucleotide spots, which are distinguished from each other by their barcode sequences, (b) providing a microwell array having microwells whose spatial arrangement corresponds to that of the DNA spots on the DNA microarray, (c) loading a solution of samples containing target nucleic acid sequences into the microwells, (d) assembling the DNA microarray to the microwell array to form micro reaction spaces in which the DNA spots are spatially separated by the microwells, (e) allowing the oligonucleotide sequences of the DNA spots to react with the target nucleic acid sequences of the samples in the micro reaction spaces to combine the sequence information of the DNA spots with the sequence information of the samples, and (f) separating the DNA microarray and the microwell array from each other to obtain reaction products including the barcode sequences.

First claim

The invention claimed is:

1. A heterologous DNA barcoding method comprising (a) providing a DNA microarray having DNA oligonucleotide spots, which are distinguished from each other by their barcode sequences, (b) providing a microwell array having microwells whose spatial arrangement corresponds to that of the DNA spots on the DNA microarray, (c) loading a solution of samples containing target nucleic acid sequences into the microwells, (d) assembling the DNA microarray to the microwell array to form micro reaction spaces in which the DNA spots are spatially separated by the microwells, (e) allowing the oligonucleotide sequences of the DNA spots to react with the target nucleic acid sequences of the samples in the micro reaction spaces to combine the sequence information of the DNA spots with the sequence information of the samples, (f) separating the DNA microarray and the microwell array from each other to obtain reaction products comprising the barcode sequences, and (g) sequencing the reaction products and analyzing them based on barcode information.

2. The heterologous DNA barcoding method according to claim 1 , wherein the oligonucleotide sequences constituting each DNA spot comprises i) a binding region binding to the target nucleic acid sequences, ii) a barcode region characterizing the spot, iii) a primer region for DNA amplification, and iv) a spacer region.

3. The heterologous DNA barcoding method according to claim 1 , wherein the DNA spots are arranged in a 1:1, 1:N or N:1 (where N is an integer greater than or equal to 2) relationship with the microwells.

4. The heterologous DNA barcoding method according to claim 1 , wherein the microwell array has a structure in which well-shaped recesses are formed on a flat substrate or a combined structure in which a flat substrate is joined to a microhole array.

5. The heterologous DNA barcoding method according to claim 1 , wherein the samples are selected from (1) DNA or RNA molecules having one or various sequences, (2) DNA microarrays having different oligonucleotide sequence information, (3) Micro/nanoparticles comprising DNA or RNA molecules thereon or therein, (4) Cells or bacteria comprising DNA or RNA molecules, (5) Viral molecules comprising DNA or RNA molecules, and (6) DNA or RNA molecules bound to proteins.

6. The heterologous DNA barcoding method according to claim 1 , wherein the sample solution comprises reagent conditions for DNA binding reaction and/or amplification reaction.

7. The heterologous DNA barcoding method according to claim 1 , wherein, in step (e), the DNAs are amplified by solid-phase PCR.

8. The heterologous DNA barcoding method according to claim 1 , wherein the oligonucleotide sequence of the DNA spots and the nucleic acid sequence of the samples are combined and amplified in one microwell environment.

9. The heterologous DNA barcoding method according to claim 1 , wherein the oligonucleotide sequence of the DNA spots and the nucleic acid sequence of the samples are combined and amplified in separate microwells, and the DNAs are amplified in other microwell containing a reagent for DNA amplification after step (f).

10. A parallel DNA analysis method comprising (a) providing a DNA microarray having DNA oligonucleotide spots, which are distinguished from each other by their barcode sequences, (b) providing a microwell array having microwells whose spatial arrangement corresponds to that of the DNA spots on the DNA microarray, (c) loading a solution of samples containing target nucleic acid sequences into the microwells, (d) assembling the DNA microarray to the microwell array to form micro reaction spaces in which the DNA spots are spatially separated by the microwells, (e) allowing the oligonucleotide sequences of the DNA spots to react with the target nucleic acid sequences of the samples in the micro reaction spaces to combine the sequence information of the DNA spots with the sequence information of the samples, (f) separating the DNA microarray and the microwell array from each other to obtain reaction products comprising the barcode sequences, (g) sequencing the reaction products, and (h) acquiring desired samples from the reaction products by selectively amplifying the DNAs of the specific spots in the DNA microarray using the barcode information of the desired samples as a primer.

11. A platform for heterologous DNA barcoding comprising:
a DNA microarray having DNA spots, which are distinguished from each other by their barcode sequences;
a microwell array assembled to the DNA microarray and having microwells whose spatial arrangement corresponds to that of the DNA spots, wherein the platform has micro reaction spaces in which the DNA spots are spatially separated from each other by the microwells; and
a solution of samples containing target nucleic acid sequences, wherein the solution of samples is loaded into the microwells in the micro reaction spaces.

12. The platform according to claim 11 , wherein the solution further contains at least one substance or composition selected from the group consisting of enzymes, primers, dNTPs, surfactants for cell lysis, compositions for DNA hybridization, and buffer compositions for PCR.

Assignees

• Seoul Nat Univ R&Db Foundation

Inventors

• Kwon Sunghoon
• Kim Junhoi
• Ryu Taehoon
• Oh Dongyoon
• Koh Jaekyung

Classifications

• C12N15/1065 Primary

  Preparation or screening of tagged libraries, e.g. tagged microorganisms by STM-mutagenesis, tagged polynucleotides, gene tags · CPC title

• C12Q1/6837

  using probe arrays or probe chips (C12Q1/6874 takes precedence) · CPC title

• C12Q2565/513

  characterised by the pattern of the arrayed oligonucleotides · CPC title

• C12Q2565/514

  characterised by the use of the arrayed oligonucleotides as identifier tags, e.g. universal addressable array, anti-tag or tag complement array · CPC title