Method for mass production of high-purity oligonucleotides

Patent metadata

Field	Value
Publication number	US-9328366-B2
Application number	US-201213524029-A
Country	US
Kind code	B2
Filing date	Jun 15, 2012
Priority date	Oct 27, 2011
Publication date	May 3, 2016
Grant date	May 3, 2016

Abstract

Provided is a method of mass-producing high-purity nucleotides including providing a sequencing substrate having a clonal library of oligonucleotides on a solid support, sequencing the clonal library, obtaining measured location data of the solid support on the sequencing substrate, mapping pixel data of a signal generated from the solid support given as a result of the sequencing with the measured location data, extracting the solid support having a desired base sequence from the sequencing substrate using the mapping result, and amplifying an oligonucleotide on the extracted solid support to replicate on a large scale.

First claim

What is claimed is:

1. A method of producing high-purity oligonucleotides, comprising the steps of:
providing a sequencing substrate having a clonal library of oligonucleotides on solid supports, wherein the solid supports are disposed in a predetermined section of the sequencing substrate;
sequencing the clonal library and generating optical or electromagnetic signals of the clonal library;
obtaining pixel data of a signal generated from each of the solid supports having a selected DNA sequence during the sequencing step;
obtaining measured location data of the solid support on the substrate by scanning the substrate with an imaging device;
mapping the pixel data with the measured location data of the solid supports,
wherein an algorithm is used in the step of mapping for tracing an exact location of each solid support;
performing the algorithm comprising:
dividing an entire domain of the substrate into a plurality of spatially small subdomains,
selecting one subdomain (i, j) among the plurality of subdomains, the one subdomain defines where a location of the pixel data and the measured location data is matched,
defining a transform function of the one subdomain by applying the matched pixel data and the measured location data,
defining an input queue having adjacent subdomains which are adjacent to the one subdomain (i, j), wherein the adjacent subdomains are (i−1, j−1), (i−1, j), (i−1, j+1), (i, j−1), (i, j+1), (i+1, j−1), (i+1, j), and (i+1, j+1),
defining a next transform function by applying any one of the adjacent subdomains to the transform function which is previously defined,
wherein the applied adjacent subdomain is added into a complete queue so that the applied adjacent subdomain is not repeatedly applied, wherein the complete queue includes the adjacent subdomains different from the input queue,
wherein an order of applying the adjacent subdomain defines a matching direction, and
determining transform functions corresponding to each subdomain of the entire domain by expanding the matching direction via repeating the step of defining the input queue and the stet of defining the next transform function,

wherein at least three mapped data are provided to trace the exact location of each solid support;
extracting the solid supports having the selected DNA sequence from the sequencing substrate by a pulsed laser beam, wherein each solid support is extracted based on the algorithm; and
amplifying an oligonucleotide on the extracted solid supports to produce a plurality of the high-purity oligonucleotides by replication.

2. The method of claim 1 , further comprising, determining a reference point by extracting the solid support located in a region of the sequencing substrate.

3. The method of claim 1 , wherein in the step of extracting, the solid supports are executed by pulsed laser ablation or radiation pressure ejection.

4. The method of claim 1 , wherein, through the step of extracting the solid supports, the solid supports move from the sequencing substrate to a receiver.

5. The method of claim 4 , wherein the receiver is configured to amplify DNA.

6. The method of claim 4 , wherein the receiver includes a plurality of wells.

7. The method of claim 1 , wherein the extracting step is executed by placing a sacrificial region between the sequencing substrate and the solid support and moving the solid supports to a receiver.

8. The method of claim 1 , wherein the extracting step is executed by moving the solid supports to an adhesive receiver.

9. The method of claim 1 , wherein the extracting step is executed by moving the solid supports to a receiver from the sequencing substrate, and by moving the solid supports to one of a plurality of wells.

10. A method of producing high-purity oligonucleotides, comprising the steps of:
providing a first sequencing substrate having a clonal library of oligonucleotides on solid supports, wherein the solid supports are disposed in a predetermined section of the first sequencing substrate;
sequencing the clonal library and generating optical or electromagnetic signals of the clonal library;
obtaining pixel data of a signal generated from each of the solid supports having a selected DNA sequence during the sequencing step;
obtaining measured location data of the solid support on the first substrate by scanning the first substrate with an imaging device;
mapping the pixel data with the measured location data of the solid supports,
wherein an algorithm is used in the step of mapping for tracing an exact location of each solid support;
performing the algorithm comprising:
dividing an entire domain of the substrate into a plurality of spatially small subdomains,
selecting one subdomain (i, j) among the plurality of subdomains, the one subdomain defines where a location of the pixel data and the measured location data is matched,
defining a transform function of the one subdomain by applying the matched pixel data and the measured location data,
defining an input queue having adjacent subdomains which are adjacent to the one subdomain (i, j), wherein the adjacent subdomains are (i−1, j−1), (i−1, j), (i−1, j+1), (i, j−1), (i, j+1), (i+1, j−1), (i+1, j), and (i+1, j+1),
defining a next transform function by applying any one of the adjacent subdomains to the transform function which is previously defined,
wherein the applied adjacent subdomain is added into a complete queue so that the applied adjacent subdomain is not repeatedly applied, wherein the complete queue includes the adjacent subdomains different from the input queue,
wherein an order of applying the adjacent subdomain defines a matching direction, and
determining transform functions corresponding to each subdomain of the entire domain by expanding the matching direction,

wherein at least three mapped data are provided to trace the exact location of each solid support;
extracting the solid supports having the selected DNA sequence from the first sequencing substrate by a pulsed laser beam, each solid support be extracted based on the algorithm, wherein the step of extracting the solid support comprises,
transferring the solid supports from the first sequencing substrate to a second substrate, and
applying the pulsed laser beam to the second substrate having the solid supports which are transferred from the first sequencing substrate; and
amplifying an oligonucleotide on the extracted solid supports to produce a plurality of the high-purity oligonucleotides by replication.

11. The method of claim 10 , wherein the second substrate has adhesion and curability.

Assignees

• Kwon Sunghoon
• Kim Hyoki
• Lee Howon
• Kim Sungsik
• Ryu Taehoon
• Snu R&Db Foundation

Inventors

• Kwon Sunghoon
• Kim Hyoki
• Lee Howon
• Kim Sungsik
• Ryu Taehoon

Classifications

• C40B60/04

  Integrated apparatus specially adapted for both screening libraries and identifying library members · CPC title

• B01J2219/00648

  by the use of solid beads · CPC title

• C40B20/02

  Identifying library members by their fixed physical location on a support or substrate · CPC title

• B01J2219/00315

  Microtiter plates · CPC title

• B01J2219/00722

  Nucleotides · CPC title