Oligonucleotide Isolation via Yeast Barcoding and Sequencing

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Solution Overview

Problem

Current array-based oligonucleotide synthesis platforms face challenges with high error rates and low yields, making it difficult to isolate specific oligonucleotides from complex mixtures without set-specific common priming sites, which limits their utility in biological and medical applications.

Innovation Solution

A method involving high-throughput production of clones, barcoding, and next-generation sequencing is used to isolate sequence-verified oligonucleotides by transforming host cells, introducing oligonucleotides into barcoder cells for site-specific recombination, and sequencing to identify and isolate correct oligonucleotides from complex mixtures.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Ease of manufacture

If array-based oligonucleotide synthesis is used, then cost is reduced (2-4 orders of magnitude lower than column synthesis), but error rate increases and yield decreases

Engineering Contradiction:

ImprovecostVSAvoiderror rate

Core Design Contradiction:

Ease of manufactureVSReliability

Solution Approach 1:

The method segments the oligonucleotide library into individual clones by transforming host cells, allowing each oligonucleotide to be isolated and verified separately. This segmentation enables error detection and correction while maintaining the cost benefits of array-based synthesis.

Inventive Principle:

Principle #1Segmentation

Solution Approach 2:

Host cells serve as intermediaries to carry and maintain the oligonucleotide library. The barcoding system acts as another intermediary, enabling identification and tracking of individual oligonucleotides through sequencing, thus resolving the reliability issue without abandoning cost-effective array synthesis.

Inventive Principle:

Principle #24Intermediary (Mediator)

2Ease of manufacture

If array-based oligonucleotide synthesis is used, then cost is reduced, but yield decreases

Engineering Contradiction:

ImprovecostVSAvoidyield

Core Design Contradiction:

Ease of manufactureVSProductivity

Solution Approach 1:

The method performs preliminary amplification of oligonucleotides in host cells before isolation, increasing the yield of each individual oligonucleotide. This preliminary action in the biological system compensates for the low initial yield of array synthesis while maintaining cost efficiency.

Inventive Principle:

Principle #10Preliminary action

Solution Approach 2:

Host cells serve as copying machines, replicating the oligonucleotides multiple times through cell division. This biological copying mechanism dramatically increases the yield of each oligonucleotide sequence without requiring additional array synthesis cycles.

Inventive Principle:

Principle #26Copying

3Productivity

If complex pools of oligonucleotides are delivered, then high-throughput production is achieved, but retrieval of specific oligonucleotides becomes imperfect and requires set-specific common priming sites

Engineering Contradiction:

Improvehigh-throughput productionVSAvoidretrieval of specific oligonucleotides

Core Design Contradiction:

ProductivityVSEase of operation

Solution Approach 1:

Unique barcodes serve as intermediaries between the oligonucleotide sequence and its physical location in the library. Each barcode is linked to a specific oligonucleotide through host cell transformation, enabling precise retrieval without requiring common priming sites. The barcode acts as a unique identifier that simplifies the retrieval process.

Inventive Principle:

Principle #24Intermediary (Mediator)

Solution Approach 2:

The method replaces the mechanical/chemical approach of using common priming sites for retrieval with a biological information system based on barcoding and sequencing. This substitution enables perfect retrieval of any individual oligonucleotide from the complex pool without requiring shared sequence elements.

Inventive Principle:

Principle #28Mechanics substitution (Replace mechanical system)

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 method enables efficient isolation of sequence-verified oligonucleotides with reduced errors, allowing for their use in various applications such as gene synthesis, CRISPR interference, and metagenomics, while simplifying the retrieval of specific oligonucleotides from complex libraries.

Implementation Method 1

translocating the oligonucleotide to a position adjacent to the barcode sequence of the barcoder cell using a site-specific recombinase system, wherein site-specific recombination with the recombination target site of the barcoder cell generates a nucleic acid comprising a barcode-oligonucleotide fusion sequence

Methodology Applied

Scientific EffectSite-specific recombination:

Data Source

PatentUS11505794B2Scalable method for isolation and sequence-verification of oligonucleotides from complex libraries

Publication Date: 2022.11.22 THE BOARD OF TRUSTEES OF THE LELAND STANFORD JUNIOR UNIV

AI summary

A novel method for preparing sequence-verified oligonucleotides is disclosed. In particular, the invention relates to a simple, affordable, and scalable method that combines high-throughput mating of yeast clones, a unique selectable system for combining DNA sequences in yeast, and next-generation sequencing. This method allows sequence-verified oligonucleotides to be readily isolated from complex libraries.