Synthesis of sequence-verified nucleic acids

a nucleic acid and sequence verification technology, applied in the field of synthetic nucleic acids, can solve the problems of not finding such widespread application, and achieve the effects of high error rate and cost, high quality, and high impact of oligonucleotide input error ra

Inactive Publication Date: 2010-08-26
SYNTHETIC GENOMICS INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0277]The advantage of the present invention is to be seen in a method for efficient and cost-effective production of sequence-verified nucleic acid fragments. Sequence-verified nucleic acid fragments are the basis for a broad spectrum of applications.
[0279]Production of so-called “perfect parts” (the sequence-verified nucleic acid fragments themselves)
[0280]a perfect part is a nucleic acid available for an application with a known sequence
[0281]Creation of repositories of so-called bio-bricks that resemble generic building blocks for molecular or synthetic biology or biotechnology applications or genetic engineering. These biobricks can be stored away e.g. in microtiter format or any other suitable format and are ready-to-use on demand
[0282]Production of bead-arrays by cost-effective production of large variety of short to long sequence-verified nucleic acid fragments. The produced fragments are reformatted in such a way that they are affixed to beads. In one embodiment an amplification reaction is carried out before the fragments are affixed to beads
[0283]Production of microarrays by cost-effective production of large variety of short to long sequence-verified nucleic acid fragments. The produced fragments are spotted onto a support to form a microarray

Problems solved by technology

Other methods, such as the H-phosphonate method, serve the same purpose of successive synthesis of a polymer from its subunits, but have not found such widespread application as the method according to Caruthers.

Method used

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  • Synthesis of sequence-verified nucleic acids
  • Synthesis of sequence-verified nucleic acids
  • Synthesis of sequence-verified nucleic acids

Examples

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Effect test

example 1

7.1 Example 1

Preparative Next Generation Sequencing

High Parallel In Vitro Cloning and Sequencing for Gene Synthesis and Library Preparation

7.1.1 Summary

[0412]The major cost factors of synthetic gene production are oligonucleotides as starting material for gene assembly, and the subsequent screening and selection of correct parts from a mixture of correct and defective gene fragments resulting from errors in oligonucleotide synthesis(1). While microarrays and especially photo-programmable microfluidic chips(2,3) represent an effective tool to decrease the cost for oligonucleotide production by orders of magnitude), the overall production cost for synthetic genes remains high due to the need for cloning and sequencing post-synthesis. Here we describe a proof of concept for a high throughput retrieval of clonal DNA with known sequence from a next generation sequencing (NGS) platform(5). This technology will reduce efforts for quality control, screening and selection of DNA significantl...

example 2

7.2 Example 2

[0439]Analysis of sequence data of mega cloned DNA fragments.

[0440]319 clonal beads from a picotiter plate (PTP) of a 454 sequence instrument were picked. Each bead carried a different sequence from a PTP. The bead-associated nucleic acids were amplified and pooled. The pooled nucleic acid sequences were sequenced on an Illumina GAII instrument to analyse the sequence verification.

[0441]The results are shown in FIG. 10.

[0442]For 304 beads the correct sequence was found with at least 20× coverage which should be regarded as solid proof that the correct sequence has indeed been picked. With a sequence length of 40 by fragment, the overall DNA length from this run was at least 1260 by error-free DNA.

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Abstract

The invention relates to methods and devices for preparing synthetic nucleic acids.

Description

[0001]The invention relates to methods and devices for preparing synthetic nucleic acids.1. INTRODUCTION[0002]There is a high demand for synthetic nucleic acids in molecular biology and biomedical research and development. Synthetic nucleic acids (DNA, RNA or their analogues) are mainly prepared using column-based synthesizers.[0003]Particularly important and widespread applications for synthetic nucleic acid polymers are primers for the polymerase chain reaction (PCR) (Critical Reviews in Biochemistry and Molecular Biology 26 (3 / 4), 301-334, 1991) and the sequencing method according to Sanger (Proc. Nat. Acad. Sci. 74, 5463-5467, 1977).[0004]Synthetic DNA also has a role in the preparation of synthetic genes. Methods of gene synthesis are described for example in U.S. Pat. No. 6,586,211 B1, in PCT / EP2004 / 013131, in WO 00 / 13017 A2, in S. Rayner et al., PCR Methods and Applications 8 (7), 741-747, 1998, in WO 90 / 00626 A1, in EP 385 410 A2, in WO 94 / 12632 A1, in WO 95 / 17413 A1, in EP ...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C40B20/08C12M1/34C12Q1/68
CPCC12Q1/6869C12N15/1093
Inventor STAEHLER, PEER F.CARAPITO, RAPHAELSTAEHLER, CORD F.MATZAS, MARKLEONARD, JACK T.JAEGER, JOACHIMBEIER, MARKUS
Owner SYNTHETIC GENOMICS INC
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