Asymmetric Splint Nucleic Acid for Efficient Cyclization
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Solution Overview
Problem
Existing library construction technologies for DNA nanoball sequencing result in low cyclization efficiency of single-stranded nucleic acid molecules, leading to imbalanced base separation and reduced accuracy in DNA quantification and SNP determination due to improper choice of splint nucleic acid molecules causing linear complexes and renatured double-stranded structures.
Innovation Solution
A splint nucleic acid molecule with asymmetric complementary regions of different lengths, allowing for two-stage gradient annealing, is used to cyclize single-stranded nucleic acid molecules, combined with a thermostable ligase to prevent intermolecular ligation and enhance self-cyclization efficiency.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If a conventional splint nucleic acid molecule with symmetric complementary regions is used, then the annealing process is simple, but the cyclization efficiency is low due to formation of linear complexes and renatured double-stranded structures
Solution Approach 1:
The splint nucleic acid molecule is designed with asymmetric complementary regions where the first complementary region has a different length than the second complementary region. This asymmetry creates different annealing temperatures for each region, preventing symmetric binding that would lead to linear complexes or renatured double-stranded structures, thereby significantly improving cyclization efficiency to 90% or above.
2Measurement precision
If the first and second complementary regions have the same length, then the splint nucleic acid molecule structure is simple, but intermolecular ligation occurs causing base separation and reduced accuracy
Solution Approach 1:
By making the first complementary region length different from the second complementary region length, the splint nucleic acid molecule prevents symmetric intermolecular ligation. The asymmetric design ensures that the 5' end and 3' end of the single-stranded nucleic acid molecule bind at different temperatures, eliminating the formation of linear complexes and renatured double-stranded structures that cause base separation, thus achieving 90% or above cyclization efficiency and eliminating visible base separation.
3Productivity
If a single annealing temperature is used, then the annealing process is simple, but both complementary regions cannot anneal properly due to different length requirements
Solution Approach 1:
The annealing process is divided into two distinct stages with different temperature ranges. The first complementary region anneals at a first temperature range suitable for its length, followed by the second complementary region annealing at a second temperature range suitable for its length. This periodic, two-stage annealing approach allows proper binding of both asymmetric regions, achieving 90% or above self-ligation efficiency.
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 significantly improves the intramolecular self-ligation efficiency of single-stranded nucleic acid molecules, reducing base separation rates to 0.5% or below, ensuring high accuracy in DNA quantification and SNP determination.
Implementation Method 1
the first complementary region and the second complementary region can be formed via annealing at different temperatures
Implementation Method 2
mediated by splint nucleic acid molecules and ligases
Data Source
AI summary
Provided in the present invention are a splint nucleic acid molecule for cyclizing a single-stranded nucleic acid molecule and an application therefor. The splint nucleic acid, molecule is composed of a 5′ terminal fragment and a 3′ terminal fragment, the 5′ terminal fragment being adapted to forming a first complementary region with a 5′ terminal of the single-stranded nucleic acid molecule, and the 3′ terminal fragment being suited to forming a second complementary region with a 3′ terminal of the single-stranded nucleic acid molecule, the length of the first complementary region and the second complementary region being different.