Probe Pool Synthesis via Nicking Enzyme Cleavage

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Solution Overview

Problem

Current methods for preparing pools of probes for multiplex nucleic acid analysis are inefficient and costly, particularly when scaling up for large numbers of samples, as they require costly chemical synthesis and lack effective methods for amplifying and separating probes from precursors and by-products.

Innovation Solution

The development of methods to synthesize oligonucleotide precursors with common priming regions that can be amplified to generate milligram quantities of probes, using nicking restriction enzymes and affinity selection to separate processed probes from intermediates and by-products, allowing for inexpensive and efficient production of probe pools.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Productivity

If chemical synthesis is used to prepare pools of probes, then probe pools can be produced, but the process is costly and inefficient for large-scale production

Engineering Contradiction:

Improveproduction efficiencyVSAvoidmanufacturing cost

Core Design Contradiction:

ProductivityVSEase of manufacture

Solution Approach 1:

The probe synthesis process is divided into two stages: (1) chemical synthesis of short oligonucleotide precursors containing universal priming sites, and (2) enzymatic amplification of these precursors to generate full-length probe pools. This segmentation allows inexpensive precursor synthesis followed by efficient amplification, resolving the contradiction between production efficiency and manufacturing cost.

Inventive Principle:

Principle #1Segmentation

Solution Approach 2:

Universal priming sites are incorporated into the oligonucleotide precursors during chemical synthesis before the amplification step. This preliminary action enables subsequent efficient amplification of the precursors to generate large quantities of probes, making the overall process both cost-effective and scalable.

Inventive Principle:

Principle #10Preliminary action

2Quantity of substance

If probes are amplified in pools, then milligram quantities can be generated from nanogram amounts of precursor, but methods for separating probes from precursors and by-products are needed

Engineering Contradiction:

Improveprobe quantityVSAvoidseparation complexity

Core Design Contradiction:

Quantity of substanceVSDevice complexity

Solution Approach 1:

Nicking restriction enzymes are used as intermediaries to selectively cleave precursor molecules at specific recognition sites, separating them from the amplified probe products. This enzymatic mediation provides a simple and effective separation method that does not require complex purification equipment, resolving the contradiction between generating large quantities of probes and managing separation complexity.

Inventive Principle:

Principle #24Intermediary (Mediator)

3Adaptability or versatility

If common priming regions are used in precursor probes, then multiplex amplification is enabled, but specific separation of individual probes from the pool becomes challenging

Engineering Contradiction:

Improvemultiplex capabilityVSAvoidprobe specificity

Core Design Contradiction:

Adaptability or versatilityVSMeasurement precision

Solution Approach 1:

While universal priming sites are used in all precursors for multiplex amplification, each individual probe retains its unique target-specific sequence in the final product. This local differentiation allows probes to be amplified together as a pool yet remain specific to their respective targets, resolving the contradiction between multiplex capability and probe specificity.

Inventive Principle:

Principle #3Local quality

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 the cost-effective and efficient generation of large quantities of probes, facilitating the analysis of thousands of samples by allowing for inexpensive synthesis and amplification of probe pools, while effectively separating and purifying the probes from precursors and by-products.

Implementation Method 1

Both preparative priming regions have recognition sites for nicking restriction enzymes arranged so that after amplification the final product can be cleaved from the double stranded amplification product

Methodology Applied

Scientific EffectNicking enzyme recognition and cleavage: Enzyme

Implementation Method 2

All ends of the double stranded product are labeled with an affinity selection reagent such as biotin so that the processed probe can be separated from the intermediates and byproducts

Methodology Applied

Scientific EffectAffinity selection: Adsorption

Implementation Method 3

The two precursors are hybridized through a unique complementary region, for example the tag region or one of the target homology regions

Methodology Applied

Scientific EffectOligonucleotide hybridization:

Implementation Method 4

the 3' end of each is extended using the other as template

Methodology Applied

Scientific EffectDNA synthesis:

Data Source

PatentUS11274327B2Methods for synthesizing pools of probes

Publication Date: 2022.03.15 AFFYMETRIX INC

AI summary

Compositions, methods and kits are disclosed for synthesizing and amplifying pools of probes using precursor oligonucleotides. In some aspects the precursor is amplified and nicking enzymes are used to separate the full length probes from the amplification products. The methods enable the preparation of single stranded DNA probes of defined sequence and length that are suitable for use in target detection assays.