G Tail Length Measurement Using Chemiluminescent Probes

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Solution Overview

Problem

Current methods for measuring the length of telomere single-stranded overhangs (G tails) are cumbersome, requiring denaturation and radioactive labels, leading to low sensitivity and long processing times, making them unsuitable for real-time monitoring and high-throughput analysis.

Innovation Solution

A method using chemiluminescence intensity to measure G tail length without denaturing chromosomal DNA, employing a labeled DNA probe complementary to telomere repeat sequences and exonuclease treatment to enhance signal-to-noise ratio, allowing for rapid and sensitive quantification.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Measurement precision

If conventional methods using radioactive labels and denaturation are used to measure G tail length, then measurement capability is achieved, but processing time increases and sensitivity decreases

Engineering Contradiction:

ImproveG tail length measurement sensitivityVSAvoidprocessing time

Core Design Contradiction:

Measurement precisionVSLoss of time

Solution Approach 1:

The invention extracts and removes the denaturation step from the conventional measurement protocol. By designing probes that can hybridize to G tails in native, double-stranded telomeric DNA context, the method eliminates the time-consuming denaturation and reannealing steps while maintaining measurement capability through specific probe design that targets the single-stranded overhang region

Inventive Principle:

Principle #2Taking out (Extraction)

Solution Approach 2:

The invention replaces the mechanical/chemical denaturation process with a biochemical recognition mechanism. The labeled probe specifically recognizes and binds to the G tail sequence in its native state through complementary base pairing, substituting the physical denaturation step with a selective molecular recognition event that occurs under physiological conditions

Inventive Principle:

Principle #28Mechanics substitution (Replace mechanical system)

2Measurement precision

If conventional methods with denaturation and radioactive labels are used, then G tail measurement is possible, but operational complexity increases

Engineering Contradiction:

ImproveG tail length measurement capabilityVSAvoidmeasurement procedure simplicity

Core Design Contradiction:

Measurement precisionVSEase of operation

Solution Approach 1:

The invention extracts and removes multiple complex steps from the conventional protocol: denaturation, radioactive labeling procedures, and extensive washing steps. The simplified method uses non-radioactive fluorescent labels that can be directly incorporated into probes, eliminating the need for separate labeling steps and reducing operational complexity

Inventive Principle:

Principle #2Taking out (Extraction)

Solution Approach 2:

The invention changes the detection parameter from radioactive signal to fluorescent signal. This parameter change enables the use of non-radioactive fluorescent labels that are easier to handle, require no special safety protocols, and can be detected directly without complex imaging systems, thereby simplifying the overall measurement procedure

Inventive Principle:

Principle #35Parameter changes

3Measurement precision

If conventional methods are used for G tail measurement, then measurement is achieved, but throughput decreases

Engineering Contradiction:

ImproveG tail length measurement accuracyVSAvoidanalysis throughput

Core Design Contradiction:

Measurement precisionVSProductivity

Solution Approach 1:

The invention performs preliminary action by pre-designing and synthesizing fluorescently labeled probes with sequences complementary to telomeric repeat units. These probes are prepared in advance with optimized hybridization characteristics, allowing direct application to samples without preliminary denaturation or labeling steps, thereby enabling high-throughput processing

Inventive Principle:

Principle #10Preliminary action

Solution Approach 2:

The invention replaces the time-consuming radioactive detection system with a fluorescent detection system that can be read directly in standard microplate readers. This substitution eliminates the need for film exposure, phosphorimaging, and radioactivity safety procedures, enabling rapid sequential processing of multiple samples and significantly increasing throughput

Inventive Principle:

Principle #28Mechanics substitution (Replace mechanical system)

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

Enables specific and rapid measurement of G tail length with high sensitivity, reducing processing time and eliminating the need for radioactive labels, facilitating real-time monitoring and high-throughput analysis.

Implementation Method 1

hybridizing a G tail of nondenatured chromosomal DNA in a sample with a labeled DNA probe having a sequence complementary to a telomere repeat sequence

Methodology Applied

Scientific EffectHybridization:

Implementation Method 2

measuring chemiluminescence from the hybridized DNA probe

Methodology Applied

Scientific EffectChemiluminescence: Chemiluminescence

Data Source

PatentUS9932627B2Method for determination of the length of the G-tail sequence and kit for the method

Publication Date: 2018.04.03 HIROSHIMA UNIVERSITY

AI summary

A method of measuring the length of a G tail sequence, characterized by hybridizing the G tail of an nondenatured chromosomal DNA in a sample with a labeled DNA probe having a sequence complementary to the telomere repeat sequence, measuring chemiluminescence from the hybridized DNA probe, and determining the length of the G tail sequence from the measured value, and a kit used for use in such a method.