Telomerase Detection Primer Set Prevents Dimer Formation
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Solution Overview
Problem
Conventional quantitative polymerase chain reaction (qPCR) methods fail to detect telomerase activity in single living cells and are prone to primer dimer formation, limiting their effectiveness in telomerase activity assays.
Innovation Solution
A primer set comprising specific upstream and downstream primers, optimized to prevent primer dimer formation, is used in conjunction with a two-reaction protocol involving telomerase extension and qPCR amplification, allowing for the detection of telomerase activity in single cells by forming hairpin structures and using fluorescent reporter groups for real-time quantitation.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If conventional qPCR methods are used, then telomerase activity can be detected, but primer dimer formation occurs and single cell detection capability is lost
Solution Approach 1:
The patent applies local quality by designing primers with specific local structural features - the downstream primer forms a hairpin structure at its 3' end, and the upstream primer has a specific sequence composition. These localized structural modifications prevent primer dimer formation while maintaining amplification capability, directly addressing the harmful effect without sacrificing detection sensitivity
Solution Approach 2:
The patent changes the sequence parameters of the primers to resolve the contradiction. Specifically, the downstream primer sequence is designed to form a hairpin structure, and the upstream primer sequence is optimized to prevent cross-pairing. These parameter changes in primer sequences eliminate primer dimer formation while preserving telomerase activity detection capability
2Measurement precision
If conventional qPCR methods are used, then telomerase activity can be detected, but single living cell detection is not achieved
Solution Approach 1:
The patent segments the detection process into two distinct reactions: telomerase extension reaction and qPCR amplification reaction. This segmentation allows the telomerase extension to occur first with the upstream primer, creating a template specific to telomerase activity, followed by selective amplification with the downstream primer. This segmentation enables single cell detection by ensuring that only telomerase-positive cells produce the specific template for amplification
Solution Approach 2:
The patent applies preliminary action by performing telomerase extension before qPCR amplification. The upstream primer first binds to the telomerase template and extends it, creating a specific product that indicates telomerase activity. Only after this preliminary extension does the qPCR amplification occur with the downstream primer. This preliminary action ensures that the amplification step only amplifies telomerase-positive samples, enabling single cell detection without requiring complex sorting or enrichment procedures
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 effectively prevents primer dimer formation and enables sensitive detection of telomerase activity down to single molecule levels, offering improved linearity, reproducibility, and sensitivity compared to existing methods.
Implementation Method 1
The fluorescent reporter group is selected from the group consisting of FAM (fluorescein amidite), BODIPY (boron-dipyrromethene), and TAMRA (tetramethylrhodamine)
Data Source
AI summary
A primer set for detecting telomerase activity, the primer set including a first primer set or a second primer set. The first primer set includes: an upstream primer selected from MTS; and a downstream primer selected from the group consisting of ACX-M4, Beacon ACX62-2C, and Beacon ACX62-10. The second primer set includes: an upstream primer selected from STS or CTS; and a downstream primer selected from the group consisting of ACX, CXT, ACX-M4, Beacon ACX62-2C, or Beacon ACX62-10. The sequences of the primers ACX, CXT, ACX-M4, Beacon ACX62-2C, Beacon ACX62-10, STS, CTS and MTS are shown as SEQ ID NOs: 1 to 8, respectively.