Deprotectable Primers for PCR-Generated DNA Sticky Ends

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Solution Overview

Problem

Existing methods for producing double-stranded DNA with sticky ends are inefficient and costly, often requiring exonucleases that can be site-specific and difficult to quantify, leading to low ligation efficiency.

Innovation Solution

A primer with a decomposable protecting group introduced into the sugar moiety of nucleosides is used, allowing for the formation of double-stranded DNA with sticky ends through PCR, followed by deprotection to create 3′-recessed and 5′-protruding ends without the need for restriction enzymes.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Reliability

If exonuclease is used to form sticky ends, then site specificity is achieved, but production cost increases and ligation efficiency decreases

Engineering Contradiction:

Improvesite specificityVSAvoidproduction cost

Core Design Contradiction:

ReliabilityVSEase of manufacture

Solution Approach 1:

The patent extracts the sticky end formation function from the exonuclease enzyme system and implements it through a chemical protecting group that is removed by acid treatment. This eliminates the need for expensive exonuclease enzymes while maintaining the ability to generate 3'-recessed ends with high specificity through the designed protecting group removal mechanism.

Inventive Principle:

Principle #2Taking out (Extraction)

Solution Approach 2:

The patent replaces the biological enzyme-based system (exonuclease) with a chemical system (acid-treated protecting group). This substitution eliminates the need for protein enzymes, reduces production costs, and simplifies the process while achieving the same functional outcome of generating sticky ends.

Inventive Principle:

Principle #28Mechanics substitution (Replace mechanical system)

2Reliability

If exonuclease is used to form sticky ends, then site specificity is achieved, but ligation efficiency decreases

Engineering Contradiction:

Improvesite specificityVSAvoidligation efficiency

Core Design Contradiction:

ReliabilityVSProductivity

Solution Approach 1:

Inventive Principle:

Principle #2Taking out (Extraction)

Solution Approach 2:

The protecting group is designed to automatically remove itself through acid treatment after serving its purpose of blocking the 3' end during PCR. This self-removing mechanism eliminates the need for separate enzymatic steps and improves ligation efficiency by ensuring clean, ready-to-ligate ends are formed directly during the PCR process.

Inventive Principle:

Principle #25Self-service

3Reliability

If protecting group is introduced into base, then termination efficiency is improved, but production complexity increases

Engineering Contradiction:

Improvetermination efficiencyVSAvoidproduction complexity

Core Design Contradiction:

ReliabilityVSDevice complexity

Solution Approach 1:

The patent applies local quality by placing the protecting group specifically at the 3' end position of the primer where it is needed for termination, rather than modifying all bases uniformly. This localized approach maintains termination efficiency while simplifying the synthesis process compared to comprehensive base modification.

Inventive Principle:

Principle #3Local quality

Solution Approach 2:

The patent changes the chemical parameter of the 3' end base by introducing a specific protecting group that can be removed by acid treatment. This parameter change enables controlled termination during PCR while the acid-labile nature of the protecting group allows for straightforward removal afterward, reducing production complexity.

Inventive Principle:

Principle #35Parameter changes

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

The method achieves high termination efficiency and low production costs, enabling flexible ligation of DNA sequences without enzyme use, enhancing the formation of sticky ends for seamless cloning.

Implementation Method 1

followed by deprotection to create 3′-recessed and 5′-protruding ends

Methodology Applied

Scientific EffectDeprotection:

Implementation Method 2

using a primer that binds complementarily to the template DNA

Methodology Applied

Scientific EffectComplementary base pairing:

Implementation Method 3

the template DNA is amplified by repeating a cycle of thermal denaturation and annealing multiple times

Methodology Applied

Scientific EffectPolymerization:

Data Source

PatentUS12448637B2Primer, device for producing double-stranded DNA using primer, and method for producing double-stranded DNA using primer

Publication Date: 2025.10.21 THE JAPAN SCI & TECH AGENCY

AI summary

A primer for amplifying a nucleic acid having a structure represented by the formula (1):where B represents a base, R1 represents a decomposable protecting group, and R2 represents hydrogen or a hydroxyl group, and the symbol * represents a bond to a sugar of an adjacent nucleotide. A device for producing double-stranded DNA, includes: a forward primer and a reverse primer, having a structure represented by formula (1); a PCR device for forming double-stranded DNA with 3′-recessed ends by performing multiple cycles of PCR by using a template DNA as a template; and a photoirradiation unit for deprotecting R1 and forming a sticky end with a 3′-protruding end.