Dual-Tagged Primers for Nucleotide Repeat Quantification
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Solution Overview
Problem
Current high-resolution electrophoretic methods for quantifying nucleotide expansions, contractions, or deletions in genetic sequences are slow, expensive, and cumbersome, making them unsuitable for mass screening and preliminary segregation of samples in genetic studies.
Innovation Solution
A method involving amplification of target nucleic acid sequences with dual-tagged primers, followed by restriction enzyme digestion to produce variation and internal control fragments, which are then hybridized and detected on capture complexes to determine the size of nucleotide expansions, contractions, or deletions, allowing for semi-quantitative or quantitative evaluation.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If high resolution electrophoretic methods are used to quantify nucleotide expansions, then measurement precision is improved, but productivity deteriorates
Solution Approach 1:
The method segments the analysis into distinct functional components: dual-tagged primers enable specific amplification of target regions, restriction enzymes create predictable fragment patterns, and fluorescent labels provide detectable signals. This segmentation allows each component to be optimized independently while maintaining overall accuracy, enabling high-throughput screening without sacrificing measurement precision
2Measurement precision
If high resolution electrophoretic methods are used to quantify nucleotide expansions, then measurement precision is improved, but loss of time increases
Solution Approach 1:
The dual-tagged primers are designed in advance with specific restriction enzyme recognition sites and fluorescent labels incorporated into the primer sequences. This preliminary design allows the PCR amplification to directly produce fragments ready for restriction digestion and detection, eliminating multiple preparation steps and reducing overall analysis time while maintaining quantification accuracy
3Measurement precision
If high resolution electrophoretic methods are used to quantify nucleotide expansions, then measurement precision is improved, but device complexity increases
Solution Approach 1:
The dual-tagged primer design serves multiple functions simultaneously: it amplifies the target sequence, incorporates restriction enzyme recognition sites for fragmentation, and includes fluorescent labels for detection. This multi-functionality consolidates what would otherwise require separate reagents and steps into a single universal primer design, simplifying the overall methodology while maintaining measurement precision
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 efficient and cost-effective mass screening and preliminary segregation of samples for genetic diseases associated with nucleotide repeat expansions, such as Fragile-X syndrome, by providing a rapid and accurate assessment of nucleotide repeat lengths.
Implementation Method 1
digesting the dual-tagged amplification product with a restriction enzyme to produce a variation region fragment and an internal control fragment
Implementation Method 2
hybridizing the variation region fragment to a first capture complex comprising an anti-tag sequence and a solid support, and hybridizing the internal control fragment to a second capture complex comprising an anti-tag sequence and a solid support
Data Source
AI summary
The methods and compositions of the present invention allow for the evaluation of a nucleotide expansion, contraction or deletion. In one embodiment, the present invention provides a method for detecting a nucleotide expansion, contraction or deletion comprising amplifying a target nucleic acid sequence with a primer pair, each primer comprising a target specific sequence and a differentiating tag sequence, labeling the target nucleic acid sequence to produce a dual-tagged amplification product; digesting the dual-tagged amplification product to produce an expansion region fragment and an internal control fragment; hybridizing the fragments to separate capture complexes; detecting the signals produced by the labels on the immobilized fragments and comparing the intensity of the signals to detect the nucleotide expansion, contraction or deletion region.