DNA End Detection via Rolling Circle Amplification
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Solution Overview
Problem
Current methods for detecting DNA breaks, particularly single- and double-strand breaks, are not sensitive or specific enough to identify individual DNA breaks in live or fixed cells, and existing techniques suffer from high background noise and limited sensitivity, making it difficult to assess DNA quality and detect low numbers of DNA damage accurately.
Innovation Solution
A method involving the preparation of biological material, modification of DNA ends, binding of molecules to these ends, and subsequent rolling circle amplification to enhance detection sensitivity, allowing for the direct visualization of DNA breaks using labeled oligonucleotides.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If traditional methods (TUNEL assay, immunofluorescence) are used for DNA break detection, then the detection process is simple and quick, but the sensitivity is insufficient and background noise is high, making it impossible to detect individual DNA breaks
Solution Approach 1:
The detection method is divided into multiple sequential steps: DNA end modification with biotinylated nucleotides, blocking of endogenous biotin, incubation with Streptavidin-conjugated oligonucleotides, ligation of second oligonucleotides, rolling circle amplification, and final detection with labeled oligonucleotides. Each step isolates and enhances specific signals while removing background interference.
Solution Approach 2:
The method performs preliminary blocking of endogenous biotin signals before detecting DNA breaks. This preliminary action removes background noise by preventing Streptavidin-conjugated oligonucleotides from binding to non-DNA-break biotin sites, thereby enhancing detection specificity and sensitivity for individual DNA breaks.
2Measurement precision
If direct detection methods are used, then the procedure is straightforward, but the ability to distinguish specific types of DNA lesions is limited
Solution Approach 1:
Different oligonucleotide probes with specific sequences are designed to bind to distinct DNA lesion types (single-strand breaks, double-strand breaks, apurinic/apyrimidinic sites). Each probe set targets specific local characteristics of different lesions, enabling precise differentiation and characterization of various DNA damage types through their unique binding patterns.
3Measurement precision
If high sensitivity detection is achieved through multiple amplification steps, then individual DNA breaks can be detected, but the detection time and procedural steps increase
Solution Approach 1:
Rolling circle amplification is employed to continuously amplify the DNA signal at each DNA break site. This continuous amplification process generates numerous copies of the target sequence, significantly enhancing the signal from individual DNA breaks and enabling their detection even after multiple procedural steps.
Solution Approach 2:
The method creates multiple copies of the DNA break signal through rolling circle amplification. Each DNA break site is replicated numerous times, generating abundant amplified products that can be easily detected. This copying process transforms the undetectable single-molecule signal into a detectable macroscopic signal without requiring complex real-time single-molecule detection equipment.
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 the sensitive and specific detection of single DNA breaks, improving the ability to assess DNA quality and quantify DNA damage, with enhanced sensitivity compared to traditional methods, allowing for the detection of even a single DNA break.
Implementation Method 1
incubation with the enzyme terminal deoxynucleotidyl transferase (TdT) and biotinylated deoxynucleotide triphosphates, which results in the incorporation of the labelled nucleotides to the 3'-OH ends of the breaks
Implementation Method 2
incubation with Streptavidin-conjugated oligonucleotides that are capable of hybridising with the incorporated nucleotides
Implementation Method 3
addition of a second set of oligonucleotides that hybridise with the incorporated nucleotides, and enzyme ligase to create ligatable ends
Implementation Method 4
DNA polymerase to amplify the hybridised oligonucleotides by rolling circle amplification
Implementation Method 5
fluorescently labelled oligonucleotides that hybridise with the amplification products
Data Source
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AI summary
The invention concerns the method of detection of DNA end(s) in a biological material, comprising the following steps I – III and at least one of sub-steps a-h of each of steps I - III: I. PREPARATION OF THE MATERIAL comprising a. fixation and/or permeabilization and/or lysis and/or isolation and/or fractionation and/or immobilization of the biological material, b. increasing accessibility of DNA end(s), c. blocking nonspecific binding site(s) for molecules type 2-6, in the biological material; II. PROCESSING OF DNA END(S) comprising d. ): modification of DNA end(s) by chemical or physical processing followed by binding molecules type 1 to the DNA end(s) by catalytic or noncatalytic means; blocking nonspecific binding site(s) for molecules type 2-6 in the biological material; III. RECOGNITION AND DETECTION OF THE MODIFIED DNA END(S): incubation of the biological material from step II with at least two molecules type 2 and 3 which bind to the molecules type 1 in a manner that allows steps leading to rolling circle amplification (RCA) reactions, g. detection of DNA end(s) by: i. optionally contacting suitable molecules type 4 and/or 5 with molecules type 2 and 3, wherein the molecules type 4 and/or 5 are conjugated with the oligonucleotides type 1, ii. adding oligonucleotides type 2 and enzyme ligase to allow hybridization of said added oligonucleotides type 2 to the oligonucleotides type 1 already linked to molecules type 4 and/or 5, or to molecules type 2 and 3 if they are linked to oligonucleotides type 1, and subsequently performing DNA ligation of oligonucleotides type 2, iii. performing amplification by adding enzyme polymerase and a solution of nucleotides to allow rolling circle amplification (RCA) reactions, and molecules type 6 to allow subsequent hybridization of molecules type 6 to thus obtained product of RCA reactions, h. detection of molecules type 6; wherein when more than one sub-step a – c of step I is performed then they may occur in any order. The invention concerns also use of rolling circle replication for marking the presence and position of single DNA end(s) and use above-mentioned method for detection of DNA end(s) in a biological material.