DNA Amplification Using ssNA to Suppress Inhibitory Structures
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Solution Overview
Problem
Existing DNA amplification methods face challenges with incomplete and low-yield amplification of complex DNA templates due to inhibitory regions such as G-quadruplexes, Z-DNA, and H-DNA structures, particularly in large, GC-rich, or repeated sequences, leading to renaturation issues.
Innovation Solution
Incorporating single-stranded nucleic acid (ssNA) molecules that anneal to inhibitory regions during DNA amplification to stabilize the template, preventing renaturation and ensuring complete and faithful amplification.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If ssDNA is used as template for DNA amplification, then amplification can proceed, but ssDNA renaturation forms stable structures (G-quadruplexes, Z-DNA, H-DNA) that inhibit polymerase access and reduce amplification yield
Solution Approach 1:
A single-stranded nucleic acid molecule (ssNA) is introduced as an intermediary that selectively anneals to inhibitory regions (G-quadruplexes, Z-DNA, H-DNA structures) on the ssDNA template. This ssNA acts as a mediator that prevents the formation of stable secondary structures by occupying the inhibitory regions, thereby maintaining the template in an accessible state for polymerase while not interfering with the amplification process. The ssNA can be RNA or DNA, and may be modified to prevent extension by polymerase.
Solution Approach 2:
The invention changes the chemical composition and structural parameters of the reaction system by introducing ssNA molecules with specific sequences that complement the inhibitory regions. This parameter change (adding a new molecular component with specific binding properties) transforms the thermal and structural dynamics of the ssDNA template, preventing renaturation into stable secondary structures during the amplification process.
2Adaptability or versatility
If DNA amplification is performed on complex sequences (large constructs, GC-rich regions, repeated sequences, homopolymer regions), then target amplification is achieved, but inhibitory structures form that prevent complete and faithful amplification
Solution Approach 1:
The ssNA serves as a specialized intermediary that recognizes and binds to specific complex sequence features (GC-rich regions, repeated sequences, homopolymer regions) that form inhibitory structures. By annealing to these problematic regions, the ssNA prevents them from forming stable secondary structures, allowing the polymerase to traverse these complex regions faithfully and complete the amplification of difficult-to-amplify sequences.
3Ease of operation
If denaturation step is applied to convert dsDNA to ssDNA, then template accessibility is improved, but ssDNA instability causes partial renaturation and formation of inaccessible structures
Solution Approach 1:
The ssNA molecule acts as a stabilizing intermediary that binds to the denatured ssDNA template during the amplification process. This intermediary prevents the ssDNA from renaturing into stable secondary structures by occupying the inhibitory regions, thereby maintaining the denatured, accessible state of the template throughout the reaction without requiring continuous high-temperature denaturation conditions.
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
Enhances the amplification of complex DNA templates by reducing inhibitory structure formation, improving yield and reproducibility, and producing high-quality amplified products.
Implementation Method 1
wherein the at least one type of ssNA molecule anneals to the at least one inhibitory region
Data Source
Figure 1A~1B
Figure 2
Figure 3
AI summary
The present invention relates to methods of amplifying DNA comprising: a) providing a DNA template molecule comprising at least one inhibitory region; and b) amplifying the DNA template molecule to generate an amplified DNA product, wherein the DNA template molecule is amplified in the presence of at least one type of single-stranded nucleic acid (ssNA) molecule, which cannot be extended by a polymerase, and wherein the at least one type of ssNA molecule anneals to the at least one inhibitory region and improves the amplification. The ssNA molecule may be random oligonucleotides and the inhibitory regions may be a homopolymeric region, e.g. polyA, a region with a high GC content, a repeated sequence; or a sequence that is prone to forming an inhibitory structure. The invention also relates to adaptor-ligated DNA products that may be produced by the methods.