5' Hairpin Adaptor Ligation for ssDNA-Specific Sequencing
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Solution Overview
Problem
Current high-throughput sequencing technologies struggle to specifically distinguish and sequence single-stranded DNA (ssDNA) from double-stranded DNA (dsDNA) in samples, necessitating improved library preparation methods.
Innovation Solution
A 5' hairpin adaptor with a specific structure is used for ssDNA-specific sequencing, involving a 5' end or 3' end hydroxyl group, complementary stem regions, and a loop region, along with a DNA polymerase lacking exonuclease activity for precise ligation and amplification, enabling the construction of a Liss-seq library.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If conventional library preparation methods are used, then sequencing can be performed on DNA samples, but single-stranded DNA cannot be specifically distinguished from double-stranded DNA
Solution Approach 1:
The library preparation process is segmented into distinct steps: dsDNA fragmentation, ssDNA enrichment, adapter ligation, and sequencing. This segmentation allows specific targeting of ssDNA molecules while excluding dsDNA, achieving precise measurement without excessive overall complexity
Solution Approach 2:
An adapter sequence serves as an intermediary element that specifically binds to ssDNA molecules. This adapter acts as a mediator that enables the sequencing system to specifically identify and sequence ssDNA while ignoring dsDNA, resolving the specificity issue
2Measurement precision
If existing sequencing methods are applied, then DNA sequencing is achieved, but ssDNA-specific sequencing sensitivity is insufficient
Solution Approach 1:
The method performs preliminary enrichment of ssDNA molecules before the main sequencing process. By pre-concentrating and purifying ssDNA from mixed samples, the sensitivity of subsequent sequencing is dramatically improved while maximizing recovery of the target substance
Solution Approach 2:
The protocol utilizes parameter changes in DNA structure (single-stranded vs. double-stranded conformation) to differentiate and enrich ssDNA. By exploiting physical-chemical parameter differences, the method achieves high sensitivity detection and maximum recovery efficiency
3Reliability
If traditional library preparation is used, then sequencing libraries can be constructed, but non-specific ligation occurs between adapters and dsDNA
Solution Approach 1:
The method applies preliminary anti-action by treating dsDNA with exonucleases before adapter ligation. This pre-treatment prevents dsDNA from participating in non-specific ligation reactions, ensuring high reliability of adapter binding while maintaining simple library construction protocols
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 achieves highly specific, sensitive, and stable sequencing of ssDNA, ensuring efficient library construction and high-throughput sequencing, with minimal non-specific ligation and bias, suitable for various sample types.
Implementation Method 1
the first stem region and the second stem region are complementary to each other and connect to each other by hydrogen bonds through corresponding relationship of different bases to form stem regions
Implementation Method 2
a double helix structure forms through performing renaturation treatment on the stem regions
Data Source
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AI summary
Provided is a 5'-end ligation-based single-stranded DNA (ssDNA)-specific high-throughput sequencing method, relating to the technical field of biology. The present disclosure specifically comprises a method for preparing a 5'-end ligation-based ssDNA-specific sequencing (Liss-seq) library. The method comprises the following steps of treating a sample to be tested: 1) using a DNA polymerase without exonuclease activity to fill in 5' ends of double-stranded DNA (dsDNA); 2) adding a tail to the 3' end; (3) ligating a renatured 5' hairpin adaptor; a structure of the 5' hairpin adaptor being: overhang-random base region-first stem region-loop region-second stem region, and the first stem region and the second stem region forming a double strand by means of renaturation; and 4) carrying out amplification on a ligated product. The amplified product constitutes a ssDNA sequencing library.