Solid Phase Nucleic Acid Assembly via Barcode Annealing

Resolve Bottlenecks,
Find Innovative SolutionsGenerate Solutions

Solution Overview

Problem

Conventional methods struggle to assemble large nucleic acids in one step due to high error rates and require overlapping sequences, which limits scalability and efficiency, especially for repetitive sequences or those with unique nucleotide compositions.

Innovation Solution

A method for assembling nucleic acid fragments on a solid phase using end-to-end ligation, independent of sequence, where fragments are immobilized via barcodes complementary to solid phase oligos, allowing for sequential or simultaneous annealing and ligation, enabling the assembly of repetitive and complex sequences.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Reliability

If conventional methods are used to assemble large nucleic acids, then the error rate increases and assembly becomes difficult, but the process remains complex and time-consuming

Engineering Contradiction:

Improveassembly accuracyVSAvoidassembly process complexity

Core Design Contradiction:

ReliabilityVSDevice complexity

Solution Approach 1:

The nucleic acid assembly process is divided into discrete steps: (a) annealing first nucleic acid fragment to first solid phase oligo, (b) annealing second nucleic acid fragment to second solid phase oligo, (c) ligating the fragments end-to-end, and (d) cleaving the ligated nucleic acid. This segmentation allows each step to be optimized independently, improving overall reliability while reducing complexity through standardized operations.

Inventive Principle:

Principle #1Segmentation

Solution Approach 2:

Solid phase oligos serve as intermediaries that facilitate the assembly process. The barcodes on nucleic acid fragments anneal to complementary solid phase oligos, positioning fragments for precise ligation. This intermediary system eliminates the need for complex overlapping sequence design and enables sequence-independent assembly, thereby improving accuracy without increasing complexity.

Inventive Principle:

Principle #24Intermediary (Mediator)

2Adaptability or versatility

If overlapping sequences are required for assembly, then sequence-specific constraints are imposed, but this limits the ability to assemble repetitive or complex sequences

Engineering Contradiction:

Improvesequence assembly versatilityVSAvoidsequence assembly precision

Core Design Contradiction:

Adaptability or versatilityVSManufacturing precision

Solution Approach 1:

The solid phase oligo system provides universal applicability across different nucleic acid sequences. The same ligation mechanism works for repetitive sequences, unique sequences, and complex sequences without requiring sequence-specific optimization. The barcodes and solid phase oligos create a universal interface that accommodates any sequence composition, thereby improving versatility while maintaining precision through standardized annealing and ligation chemistry.

Inventive Principle:

Principle #6Universality (Multi-functionality)

3Productivity

If large nucleic acids are synthesized in one step, then production time is reduced, but the error rate increases significantly

Engineering Contradiction:

Improveassembly speedVSAvoidassembly accuracy

Core Design Contradiction:

ProductivityVSReliability

Solution Approach 1:

Nucleic acid fragments are prepared and barcoded before assembly. The barcodes are designed to anneal specifically to complementary solid phase oligos, pre-positioning the fragments for accurate ligation. This preliminary preparation ensures that when fragments are brought together, they align correctly without requiring error-prone one-step synthesis, thereby improving both productivity through parallel processing and reliability through precise positioning.

Inventive Principle:

Principle #10Preliminary action

Applied Scientific Principles

This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.

Function Achieved in This Case

This approach enables efficient assembly of large nucleic acid sequences, including repetitive ones, with improved scalability and accuracy, overcoming the limitations of conventional technologies by eliminating the need for overlapping sequences and allowing for orientation-specific assembly.

Implementation Method 1

annealing a first nucleic acid fragment to a first solid phase oligo bound to a solid phase

Methodology Applied

Scientific EffectAnnealing: Annealing

Implementation Method 2

The first bar code can bind to the first solid phase oligo, and the second bar code can bind to the second solid phase oligo. The barcodes can be completely complementary or partially complementary to the corresponding solid phase oligos.

Methodology Applied

Scientific EffectComplementary base pairing:

Implementation Method 3

ligating the first nucleic acid fragment and the second nucleic acid fragment by end-to-end ligation

Methodology Applied

Scientific EffectLigation:

Implementation Method 4

cleaving the ligated nucleic acid at a position close to the junction of the second nucleic acid fragment and the second solid phase oligo

Methodology Applied

Scientific EffectCleavage:

Data Source

PatentUS10407460B2Solid phase sequence-independent nucleic acid assembly

Publication Date: 2019.09.10 RGT UNIV OF CALIFORNIA

AI summary

Disclosed herein are methods of assembling nucleic acid fragments, such as non overlapping nucleic acid fragments, in an orientation-specific, sequence-independent way. The methods entail annealing one or more nucleic acid fragments to be assembled onto a solid phase via matching barcodes having sequences complementary to the solid phase oligos, ligating the nucleic acid fragments by end-to-end ligation, and recovering the ligated nucleic acid from the solid phase.