Chimeric DNA Detection via Segmented Probes
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Solution Overview
Problem
Conventional methods for detecting target nucleic acids are inefficient due to prolonged hybridization times, non-specific binding, and intricate probe design requirements, making it difficult to accurately detect multiple target sequences in a short time.
Innovation Solution
A method involving the use of multiple detection probes with unique base sequences and tag sequences, where chimeric DNA is synthesized through PCR using specific primers to hybridize with detection probes, reducing non-specific binding and allowing for accurate detection without the need for complex probe design or re-design.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If conventional array methods are used for detecting target nucleic acids, then detection can be performed, but the hybridization process requires a prolonged period of time
Solution Approach 1:
The detection probe is divided into two functional segments: a detection sequence for specific binding to the target nucleic acid and a tag sequence for signal generation. This segmentation allows the detection sequence to be optimized for rapid specific hybridization while the tag sequence enables efficient signal detection, thereby reducing overall hybridization time without compromising detection accuracy
Solution Approach 2:
A tag sequence acts as an intermediary between the detection probe and the target nucleic acid. The tag sequence is amplified along with the target sequence during PCR, creating a chimeric DNA that facilitates more efficient and specific hybridization to the detection probe, thus reducing hybridization time while maintaining detection precision
2Measurement precision
If detection probes are used for detecting target nucleic acids, then detection can be performed, but non-specific binding occurs to nucleic acid sequences with homology
Solution Approach 1:
The detection probe is designed with differentiated local qualities: the detection sequence provides high specificity for target binding, while the tag sequence provides universal amplification capability. This local differentiation allows the detection sequence to be optimized for maximum specificity, reducing non-specific binding to homologous sequences while maintaining detection accuracy
Solution Approach 2:
The tag sequence is incorporated into the detection probe before hybridization, and its complementary sequence is amplified during PCR to create chimeric DNA. This preliminary incorporation ensures that only specifically bound targets are amplified and detected, preventing non-specific binding signals from interfering with detection accuracy
3Object-affected harmful factors
If the length of detection probe is reduced to minimize homology, then non-specific binding decreases, but the intensity of signal label decreases
Solution Approach 1:
The probe is segmented into a detection sequence and a tag sequence. The detection sequence is kept relatively short to minimize non-specific binding, while the tag sequence is amplified during PCR to generate sufficient signal intensity. This segmentation resolves the contradiction by allowing each segment to be optimized for its specific function
Solution Approach 2:
The tag sequence enables continuous amplification of the signal during PCR, maintaining and enhancing signal intensity even when the detection probe length is reduced. This continuous amplification ensures that signal intensity is not compromised by the reduction in probe length, while still minimizing non-specific binding
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 rapid and accurate detection of target nucleic acids by suppressing non-specific binding and optimizing hybridization conditions, allowing for simultaneous detection of multiple targets with high selectivity and sensitivity.
Implementation Method 1
the detection probe fixed on the array is hybridized with the target sequence
Implementation Method 2
carrying out PCR on the sample so as to obtain chimeric DNAs
Data Source
Figure 1
Figure 2(a)~2(c)
Figure 3
AI summary
An object of the disclosure of the present specification is to provide a method for detection of a target nucleic acid which allows construction of an effective detection system of a target nucleic acid. For this purpose, in the disclosure of the present specification, a first primer 30 comprising an identification sequence 32 complementary to a target sequence 12 in a target nucleic acid and a tag addition sequence 36, and a second primer 40 having a label 42 are prepared. The first primer 30 and the second primer 40 are used for the target nucleic acid 10 in a sample to amplify a chimeric DNA 60 having a tag sequence 66 and the label 42. The chimeric DNA 60 is hybridized with a detection probe 104 on a solid phase 100 to obtain signal intensity information based on the label 42, and the target nucleic acid 10 is detected based on the signal intensity information.