Nucleic Acid Fragmentation and LNA Primer Amplification for Mutant Detection
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Solution Overview
Problem
Current nucleic acid detection methods face challenges in detecting low levels of target nucleic acids, especially in the presence of large amounts of non-target nucleic acids, which is crucial for early cancer diagnosis and prognosis, as they often fail to differentiate between mutant and wild-type sequences effectively.
Innovation Solution
The method involves fragmenting nucleic acids using restriction enzymes to distinguish between target and non-target nucleic acids, followed by amplification with mutation-specific primers, particularly locked nucleic acid (LNA) primers that enhance specificity and sensitivity, allowing for the detection of mutant sequences even at very low percentages.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If amplification methods such as PCR are used to detect a few copies of mutant nucleic acid, then detection sensitivity is improved, but the abundance of normal nucleic acid interferes with detection
Solution Approach 1:
The nucleic acid sample is divided into fragments by restriction enzyme digestion before amplification. This segmentation reduces the complexity of the sample and separates target sequences from interfering normal nucleic acid, allowing more effective detection of mutant sequences at low concentrations
Solution Approach 2:
Restriction enzyme digestion is performed as a preliminary step before PCR amplification. This pre-treatment selectively degrades or modifies normal nucleic acid sequences while preserving target mutant sequences, reducing interference before the detection process begins
2Quantity of substance
If standard PCR amplification is used, then amplification of target nucleic acid is achieved, but specificity is reduced due to inability to distinguish mutant from wild-type sequences
Solution Approach 1:
Locked nucleic acid (LNA) primers are introduced as intermediary molecules that bridge the gap between standard PCR amplification and mutant-specific detection. The LNA primers contain sequences that are complementary only to mutant nucleic acid, providing both amplification capability and mutant-specific recognition in a single reagent
Solution Approach 2:
The primer structure combines standard nucleotide sequences with locked nucleic acid modifications to create a composite primer molecule. This composite structure provides both the amplification functionality of standard primers and the enhanced specificity of LNA for detecting mutant sequences
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 approach significantly increases the detection sensitivity for mutant nucleic acids, enabling the identification of mutant sequences at levels as low as 0.001% or less in the presence of excess wild-type DNA, facilitating early cancer diagnosis and prognosis.
Implementation Method 1
The method involves fragmenting nucleic acids using restriction enzymes to distinguish between target and non-target nucleic acids
Implementation Method 2
amplification with mutation-specific primers, particularly locked nucleic acid (LNA) primers that enhance specificity and sensitivity
Implementation Method 3
locked nucleic acid (LNA) primers that enhance specificity and sensitivity
Data Source
AI summary
Provided herein are methods and compositions for detection of a nucleic acid target in a sample. The methods and compositions use primer directed amplification in conjunction with nucleic acid fragmentation. The methods have high sensitivity even in the presence of a large amount of non-target nucleic acid. Also provided are oligonucleotides and kits useful in the method. Exemplary nucleic acid targets are those with mutant gene sequence such as mutant sequence of the EGFR, APC, TMPRSS2, ERG and ETV1 genes.