Method and apparatus for capturing high-purity nucleotides

Patent metadata

Field	Value
Publication number	US-12467074-B2
Application number	US-201816611190-A
Country	US
Kind code	B2
Filing date	May 14, 2018
Priority date	May 12, 2017
Publication date	Nov 11, 2025
Grant date	Nov 11, 2025

Abstract

The present invention relates to a technique for synthesizing DNA sequences in high purity at low cost. More specifically, the present invention relates to a method and apparatus for capturing high-purity nucleotides that is suitable for use in the field of genome/gene synthesis.

First claim

The invention claimed is:

1 . A method for capturing high-purity nucleic acid molecules, comprising (a) preparing nucleic acid fragments, (b) tagging the nucleic acid fragments with barcode sequences, (c) determining the location coordinates of sequence-validated ones of the nucleic acid fragments tagged with the barcode sequences, and (d) retrieving the sequence-validated fragments based on the location coordinates,
wherein the step (c) comprises: step (c-1) of parallel sequencing all or some of the sequences of the fragments tagged with the barcode sequences in the step (b) to identify the barcode sequences of the sequence-validated fragments; and step (c-2) comprising: i) immobilizing the fragments tagged with the barcode sequences in the step (b) on a support and ii) identifying the barcode sequences of the tagged fragments immobilized on the support by sequencing either simultaneously or sequentially to determine the location coordinates representing the barcode sequences identified by the sequence-validated fragments in the parallel sequencing step (c-1), wherein, in step (c-2-i), the fragments tagged with the barcode sequences are directly attached to the support; the support is a patterned with repeated immobilization sites for immobilizing the fragments; each of the immobilization sites has a pillar structure and allows for the amplification of one fragment; and at least one of the immobilization sites includes only one fragment, and wherein, in the step (c-2), the fragments tagged with the barcode sequences are amplified by rolling circle amplification (RCA).

2 . The method according to claim 1 , wherein, in the substep i), the support surface immobilized with the fragments tagged with the barcode sequences is composed of a polymer, silica, hydrogel, glass or a combination thereof.

3 . The method according to claim 1 , wherein, in the substep i), the interaction with the support surface to immobilize the fragments tagged with the barcode sequences on the support is electrostatic attraction, adsorption, protein binding, self-assembly, bonding of chemical functional groups or a combination thereof.

4 . The method according to claim 1 , wherein, in the step (c-2), the sequencing is performed by sequencing by synthesis (SBS), sequencing by ligation (SBL), ELISA or a combination thereof to determine the location coordinates representing the corresponding barcode sequences on the support.

5 . The method according to claim 1 , wherein the RCA is carried out using phi29 DNA polymerase.

6 . The method according to claim 1 , wherein the fragments tagged with the sequences immobilized on the support are in the form of molecular clones with a diameter of 30 μm or less.

7 . The method according to claim 1 , wherein, in the step (c), the location coordinates are determined by labeling the barcodes with fluorescent molecules and directly observing the fluorescence.

8 . The method according to claim 1 , wherein, in the step (a), the nucleic acid fragments are 20 to 3,000 bp in length.

9 . The method according to claim 1 , wherein the parallel sequencing is selected from massively parallel sequencing, Sanger sequencing, mass spectrometry, electrophoresis, hybridization, digital PCR, allele-specific PCR, quantitative PCR, fluorescence-based classification, and combinations thereof.

10 . The method according to claim 9 , wherein the massively parallel sequencing is selected from the group consisting of sequencing by synthesis, Ion Torrent sequencing, pyrosequencing, sequencing by ligation, nanopore sequencing, single-molecular real-time sequencing, and combinations thereof.

11 . The method according to claim 1 , wherein the barcode sequences are mixtures of two or more types of randomly or intentionally designed oligonucleotides.

12 . The method according to claim 1 , wherein the barcode sequences are 5 to 300 bp in length.

13 . The method according to claim 1 , wherein the tagging with the barcode sequences is performed by a method selected from the group consisting of PCR, emulsion PCR, ligation, and combinations thereof.

14 . The method according to claim 1 , wherein the step (b) is carried out by selectively amplifying the target fragments with primers corresponding to the barcode sequences and retrieving the amplification products.

15 . The method according to claim 14 , wherein the primers are capable of binding molecules that are selectively captured, wherein the molecules are at least one of biotin, biotin dT, biotin-TEG, Dual biotin, PC-biotin, acrylic group, thiol group, dithiol group, amine group, NHS ester, azide, hexynyl, octadiynyl dU, his tag, protein tag, polyadenine (polyA), sequencing adapters for NGS, and fixed DNA sequences of 10 nt or more.

16 . The method according to claim 1 , wherein, in the step (d), the retrieval based on the location coordinates is performed by applying energy in either a contact or non-contact manner with the application of ultrasonic waves, a pneumatic pressure, a laser or a combination thereof.

17 . The method according to claim 1 , further comprising amplifying or cleaving specific sequences such that nucleic acid molecules having desired sequences are further captured from the sequence-validated fragments retrieved in the step (d).

18 . The method according to claim 1 , wherein, the RCA reaction of step (c) is performed in a solution to form RCA products (RCPs) and the RCPs are then immobilized on the support.

19 . The method according to claim 18 , wherein the RCPs are immobilized on the support by coating a multi-branched silane compound on the support.

20 . The method according to claim 18 , wherein the RCPs are immobilized on the support by spraying the RCPs on the support.

21 . The method according to claim 1 , wherein the RCA reaction of step (c) comprises a step of forming ssDNA molecules of sense strands, wherein the ssDNA molecules are hybridized with a probe and circularized by ligation.

Assignees

• Seoul Nat Univ R&Db Foundation

Inventors

• Kwon Sung Hoon
• Kim Sung Sik
• Yeom Hui Ran
• Lee Yong Hee
• Kim Namphil
• Kim Hamin
• Lee Wonjun

Classifications

• C12Q2563/185

  Nucleic acid dedicated to use as a hidden marker/bar code, e.g. inclusion of nucleic acids to mark art objects or animals · CPC title

• C12Q2531/113

  PCR · CPC title

• C12Q1/6869

  Methods for sequencing · CPC title

• C12Q1/686

  Polymerase chain reaction [PCR] · CPC title

• C12N9/1252 Primary

  DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase · CPC title