3′ Overhang DNA Repair Without Sequence Loss

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Solution Overview

Problem

Existing methods for repairing 3′ overhangs in DNA molecules result in the loss of sequence information due to exonucleolysis, making it difficult to determine the sequence between 5′ and 3′ ends and nucleosome positioning.

Innovation Solution

A method involving the use of primers with a random target-hybridizing sequence, extended by a DNA polymerase lacking exonuclease activity, to ligate the 3′ end of extended primers to the 5′ end of the DNA fragment, thereby retaining the sequence of the 3′ overhangs.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Reliability

If a 3' to 5' exonuclease is used to resect the 3' overhang, then the overhang repair is achieved, but sequence information is lost

Engineering Contradiction:

Improveoverhang repair effectivenessVSAvoidsequence information

Core Design Contradiction:

ReliabilityVSLoss of information

Solution Approach 1:

Instead of using a 3' to 5' exonuclease to resect the 3' overhang (conventional approach), the invention uses a 5' to 3' polymerase to extend from the 5' end towards the 3' overhang, filling in the gap without removing any nucleotides. This inverted approach preserves all sequence information while achieving the same repair outcome.

Inventive Principle:

Principle #13The other way round (Inversion)

Solution Approach 2:

The invention introduces a 5' to 3' polymerase as an intermediary enzyme that mediates the repair process by synthesizing DNA to fill the gap created by the 3' overhang. This intermediary approach allows repair without the need for exonuclease-mediated removal, thereby preserving sequence information.

Inventive Principle:

Principle #24Intermediary (Mediator)

2Ease of manufacture

If exonucleolysis is used for 3' overhang repair, then the repair is completed, but the location of the original molecule end cannot be determined

Engineering Contradiction:

Improverepair process completionVSAvoidend location determination

Core Design Contradiction:

Ease of manufactureVSMeasurement precision

Solution Approach 1:

The invention inverts the conventional repair approach by using 5' to 3' polymerization instead of 3' to 5' exonuclease activity. This allows the original 3' end location to be preserved and identified, enabling accurate determination of end locations for downstream analysis such as nucleosome positioning.

Inventive Principle:

Principle #13The other way round (Inversion)

3Productivity

If a DNA polymerase with exonuclease activity is used, then the repair efficiency is improved, but sequence information is degraded

Engineering Contradiction:

Improverepair efficiencyVSAvoidsequence information

Core Design Contradiction:

ProductivityVSLoss of information

Solution Approach 1:

The invention selects a DNA polymerase with specific local qualities - it has 5' to 3' polymerase activity for efficient repair but lacks 3' to 5' exonuclease activity that would degrade sequence information. This selective enzyme choice achieves both high repair efficiency and information preservation.

Inventive Principle:

Principle #3Local quality

Solution Approach 2:

The invention changes the key parameter of polymerase selection from enzymes with exonuclease activity to enzymes without exonuclease activity. This parameter change maintains repair efficiency through 5' to 3' synthesis while eliminating the harmful information loss associated with exonuclease-mediated resection.

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 preserves the sequence information of 3′ overhangs, allowing for accurate sequencing and analysis of DNA fragments without the loss of information.

Implementation Method 1

extending one or more primers of the primer population along the DNA fragment using a DNA polymerase, thereby producing one or more extended primers annealed to the DNA fragment

Methodology Applied

Scientific EffectDNA polymerase extension: Enzyme

Implementation Method 2

ligating the 3' end of one or more extended primers to the 5' end of an extended primer or a strand of the partially double-stranded DNA fragment, thereby providing a repaired DNA fragment

Methodology Applied

Scientific EffectDNA ligation: Enzyme

Data Source

PatentUS12534757B2Methods for 3′ overhang repair

Publication Date: 2026.01.27 GUARDANT HEALTH INC

AI summary

Methods of repairing a partially double-stranded DNA fragment are provided. In some embodiments, the methods comprise (a) contacting the partially double-stranded DNA fragment with one or more primers of a primer population, wherein the partially double-stranded DNA fragment comprises a 3′ overhang and the primer population comprises a random target-hybridizing sequence; (b) extending one or more primers of the primer population along the DNA fragment using a DNA polymerase, thereby producing one or more extended primers annealed to the DNA fragment; and (c) ligating the 3′ end of one or more extended primers to the 5′ end of an extended primer or a strand of the partially double-stranded DNA fragment, thereby providing a repaired DNA fragment.