Modified Primers for Single-Strand Nucleic Acid Detection
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Solution Overview
Problem
Existing nucleic acid amplification methods, such as asymmetric PCR, suffer from inefficiencies and low signal-to-noise ratios due to the production of double-stranded products, which complicates sequence-specific probe hybridization and detection.
Innovation Solution
A novel amplification method using a modified primer with at least one modified nucleotide, followed by exonuclease treatment to generate single-stranded nucleic acid protected from degradation, allowing for increased signal-to-noise ratio through multiple probe hybridizations.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Ease of operation
If asymmetric PCR is used to produce single-stranded amplification products, then probe hybridization is facilitated, but amplification efficiency decreases due to linear rather than exponential reaction kinetics
Solution Approach 1:
The patent applies asymmetry by incorporating modified nucleotides into only one primer (creating an asymmetric primer pair), which allows the amplification to proceed efficiently while producing predominantly single-stranded products that are suitable for probe hybridization
Solution Approach 2:
The patent changes the chemical parameter of the primers by incorporating modified nucleotides (such as phosphorothioate modifications) into one primer, which alters the amplification kinetics and product structure to achieve both high efficiency and single-stranded output
2Quantity of substance
If high concentrations of probe are used in the detection mixture, then probe availability for hybridization increases, but noise increases and signal-to-noise ratio decreases
Solution Approach 1:
The patent extracts the problematic double-stranded structure by using modified primers that produce predominantly single-stranded amplification products, eliminating the need for high probe concentrations and reducing background noise
Solution Approach 2:
The modified nucleotides act as intermediaries that prevent re-annealing of complementary strands during amplification, ensuring single-stranded product formation that facilitates probe binding without requiring high probe concentrations
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 provides easy detection with enhanced signal-to-noise ratio by producing single-stranded nucleic acid products, overcoming inefficiencies of asymmetric PCR and maintaining detection efficiency.
Implementation Method 1
incubating the double stranded nucleic acid with a 5' to 3' exonuclease which hydrolyses the second strand but does not hydrolyse the amplified region of the first strand
Implementation Method 2
one of the two primers comprises at least one modified nucleotide... the 5' to 3' exonuclease is specific for double stranded nucleic acids and is capable of hydrolysing the second strand but is incapable of hydrolysing the first strand in the region of the at least one modified nucleotide
Implementation Method 3
The detection of amplified nucleic acid products may be carried out... by use of a double stranded-DNA intercalating dye... Alternatively, a semi-specific detection of product may be carried out by resolving approximate molecular weight of the product
Data Source
AI summary
A method of nucleic acid amplification involving using a first modified primer which provides protection to the amplification product from exonuclease degradation and a second primer. The method provides a double stranded nucleic acid, one strand of which is degraded by a double strand nucleic acid specific exonuclease to form a single stranded nucleic acid, which is protected from exonuclease degradation.