Rolling Circle Synthesis for Oligonucleotide Probe Production

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Solution Overview

Problem

Current methods for making nucleic acid probes for fluorescence in situ hybridization (FISH) are limited by the availability of genomic clones, high variability in hybridization efficiency, and the cost of custom-synthesized oligonucleotides, which restricts the ability to target specific genomic regions effectively.

Innovation Solution

The method involves producing long strands of nucleic acids using rolling circle synthesis from template oligonucleotides, which are then cleaved into multiple single-stranded probes with common primary sequences and diverse secondary sequences, allowing for indirect labeling and efficient hybridization to target nucleic acid sequences.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Adaptability or versatility

If clone-based FISH probes are used, then genomic regions can be visualized, but the availability is restricted by clone availability and insert size limitations

Engineering Contradiction:

Improveability to target specific genomic regionsVSAvoidavailability of genomic clones

Core Design Contradiction:

Adaptability or versatilityVSEase of manufacture

Solution Approach 1:

The patent uses rolling circle replication to generate multiple copies of the probe sequence from a single circular template DNA molecule. This copying mechanism produces long single-stranded DNA containing numerous repetitions of the probe sequence, eliminating the need for multiple genomic clones and enabling targeted visualization of specific genomic regions regardless of clone availability.

Inventive Principle:

Principle #26Copying

2Reliability

If genomic clones are used as templates, then probe production is possible, but hybridization efficiency varies highly among different preparations

Engineering Contradiction:

Improvehybridization efficiencyVSAvoidconsistency of probe preparation

Core Design Contradiction:

ReliabilityVSProductivity

Solution Approach 1:

The patent changes the fundamental parameter of probe structure by using rolling circle replication to generate long single-stranded DNA with repetitive probe sequences, rather than using traditional double-stranded genomic clone fragments. This parameter change ensures that all probes in a preparation have identical sequences and structures, eliminating the variability in hybridization efficiency observed with clone-based methods.

Inventive Principle:

Principle #35Parameter changes

3Manufacturing precision

If custom-synthesized oligonucleotides are used, then precise sequence targeting is achieved, but the cost is high

Engineering Contradiction:

Improvesequence precisionVSAvoidcost of probe production

Core Design Contradiction:

Manufacturing precisionVSQuantity of substance

Solution Approach 1:

The patent incorporates a cleavage site sequence into the circular template DNA during the preliminary design stage. This preliminary action enables subsequent enzymatic cleavage of the long single-stranded DNA into multiple individual probe molecules, allowing a single template to produce numerous probes and significantly reducing the cost compared to synthesizing each oligonucleotide separately.

Inventive Principle:

Principle #10Preliminary action

Solution Approach 2:

The rolling circle replication system is self-amplifying, automatically generating long strands of DNA with repetitive probe sequences from a single template molecule. This self-service mechanism eliminates the need for expensive custom synthesis of each probe and provides a cost-effective way to produce precise, sequence-specific probes.

Inventive Principle:

Principle #25Self-service

4Ease of operation

If probe DNA is fragmented into 150-250 bp pieces, then penetration into fixed cells is facilitated, but the probes are still limited by clone availability

Engineering Contradiction:

Improvepenetration into fixed cellsVSAvoidtargeting capability

Core Design Contradiction:

Ease of operationVSAdaptability or versatility

Solution Approach 1:

The patent segments the long single-stranded DNA produced by rolling circle replication into individual probe molecules of appropriate length (150-250 bp) through enzymatic cleavage at repeated cleavage sites. This segmentation maintains the penetration advantage of short probes while preserving the adaptability to target any genomic region by simply changing the circular template sequence, without being limited by clone availability.

Inventive Principle:

Principle #1Segmentation

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 production of numerous single-stranded nucleic acid probes with precise sequences and lengths, avoiding the need for double-stranded DNA separation and reducing labeling variability, thus enhancing the efficiency and cost-effectiveness of nucleic acid hybridization.

Implementation Method 1

producing long strands of nucleic acids using rolling circle synthesis from template oligonucleotides

Methodology Applied

Scientific EffectRolling circle synthesis:

Implementation Method 2

nucleic acids that can hybridize to nucleic acid sequences of interest

Methodology Applied

Scientific EffectHybridization:

Data Source

PatentUS10370702B2Methods of making oligonucleotide probes

Publication Date: 2019.08.06 PRESIDENT & FELLOWS OF HARVARD COLLEGE

AI summary

The present invention relates to methods of making linear nucleic acid probes using rolling circle amplification methods.