Nucleic Acid Library Construction via USER Enzyme Digestion
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Solution Overview
Problem
The existing methods for constructing libraries of single-stranded cyclic nucleic acid fragments with double adaptors on the CG sequencing platform are limited by small insert fragment sizes, lengthy construction processes, and high reagent consumption, which hinder genomic de novo sequencing and efficiency.
Innovation Solution
A method involving PCR amplification with U base sites, USER enzyme digestion to create sticky ends, controlled nick/gap translation, and direct cyclization of single-stranded nucleic acid molecules using mediating sequences, allowing for longer insert fragments without gel recovery and simplifying the construction process.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Length of moving object
If the existing library construction method on CG platform is used, then the library can be constructed, but the insert fragment length is too small (2×19-35 bp) which limits genomic de novo sequencing application
Solution Approach 1:
The library construction process is divided into multiple controlled steps: initial fragmentation, adaptor ligation, controlled nick translation to generate specific fragment lengths, and selective amplification. This segmentation allows precise control over insert fragment length distribution, producing fragments in the optimal range for CG sequencing while enabling genomic de novo sequencing applications.
2Reliability
If the existing library construction procedure is used, then adaptors can be ligated to genomic DNA fragments, but multiple enzymatic reaction and purification steps are required which takes much time and consumes many reagents
Solution Approach 1:
Multiple functions are merged into fewer steps: the controlled nick translation reaction simultaneously generates specific fragment lengths and creates sticky ends for cyclization; the single-strand cyclization step combines denaturation and cyclization; adaptor ligation is optimized to occur in fewer purification steps. This merging reduces the total number of enzymatic reactions and purification steps while maintaining ligation efficiency.
Solution Approach 2:
The method performs preliminary actions to simplify subsequent steps: initial fragmentation and adaptor ligation are optimized to create fragments with predetermined characteristics; controlled nick translation pre-generates the correct fragment lengths and sticky ends before cyclization. These preliminary actions reduce the complexity and time of later library construction steps.
3Length of moving object
If the NT technique is used to improve insert fragment length, then the length increases, but the spanning is very wide requiring gel recovery which increases operation tediousness and affects recovery efficiency
Solution Approach 1:
The controlled nick translation reaction uses optimized parameters (enzyme concentration, reaction time, temperature, dNTP composition) to precisely control the translation distance and generate insert fragments within a narrow, optimal length range. This parameter optimization eliminates the need for gel recovery by directly producing fragments of the desired size distribution, greatly simplifying the operation.
4Reliability
If the existing construction process is used, then sequencing adaptors can be ligated to genomic DNA fragments, but the initial sample amount required (3 μg) is high compared to other platforms
Solution Approach 1:
The controlled nick translation reaction uses the cyclic nucleic acid molecules themselves as templates, allowing the reaction to proceed efficiently with minimal starting material. The method is designed to be self-amplifying through the cyclization and nick translation steps, reducing the need for large initial sample amounts while ensuring library construction success.
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 increases insert fragment lengths, reduces construction time and reagent usage, and enhances the efficiency and cost-effectiveness of library construction, enabling broader application of the CG sequencing platform.
Implementation Method 1
digesting the first products with a USER enzyme to form sticky ends
Implementation Method 2
denaturing the second products to obtain single-stranded nucleic acid molecules
Implementation Method 3
cyclizing one of the single-stranded nucleic acid molecules with a mediating sequence complementary to both ends of the single-stranded nucleic acid molecule
Data Source
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AI summary
A method and reagent for constructing a nucleic acid double-joint single-strand cyclical library. The method comprises: breaking a nucleic acid into nucleic acid fragments; connecting a first linker sequence; producing by amplification a first product provided with the first linker sequence at either end, where a U nucleobase is provided on a primer sequence; using USER enzyme to cleave the first product and cyclizing to produce a gap; or, a nicking enzyme recognition sequence is also provided on the primer sequence, using the USER enzyme to cleave the first product, cyclizing and using a nicking enzyme for nicking to produce a nick; performing a restrictive nick/gap translation reaction from the nick or the gap; removing by digestion any portion that did not undergo the restrictive nick/gap translation reaction; connecting a second linker sequence; producing by amplification a second product provided with the second linker sequence at either end; denaturing the second product, and using a mediated sequence for cyclization of a single-strand nucleic acid molecule. The method allows an increase in the length of library insert fragments and obviates the need for gel extraction; the single-strand nucleic acid molecule can be cyclized directly when denatured with heat.