Single-Stranded DNA Library Preparation with Anti-Dimer Ligation
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Solution Overview
Problem
Existing methods for preparing double-stranded DNA libraries from single-stranded DNA fragments face inefficiencies in yield and produce significant amounts of adaptor dimers, which complicate sequencing and purification.
Innovation Solution
A method involving TACS ligation followed by topoisomerase I (VTopoI) ligation to minimize adaptor dimers, using a sequence with a nick and uracil or dSpacer to prevent dimer formation, and PCR to amplify the tagged DNA.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If conventional library preparation methods (e.g., TACS-T4) are used to improve library yield, then library yield increases significantly, but adaptor dimer production increases as a harmful by-product
Solution Approach 1:
The invention extracts and removes the harmful adaptor dimer by-products from the library preparation process through selective purification steps, separating them from the desired library products to resolve the contradiction between high yield and low dimer contamination
Solution Approach 2:
The invention changes the chemical parameters of the adaptor sequences by introducing specific modifications (such as different phosphorylation patterns, overhang sequences, or chemical modifications) that prevent adaptor dimers from forming while maintaining efficient ligation to target DNA, thus achieving high yield without dimer by-products
2Manufacturing precision
If adaptor dimers are removed by purification to improve sequencing quality, then sequencing accuracy improves, but the process becomes more complex and time-consuming
Solution Approach 1:
The invention performs preliminary action by designing adaptors with built-in anti-dimer features (such as asymmetric phosphorylation or specific overhang sequences) that prevent dimer formation at the source, eliminating the need for complex post-preparation purification steps and simplifying the overall workflow while maintaining high sequencing quality
3Manufacturing precision
If adaptor dimers are removed by purification, then sequencing accuracy improves, but the process requires additional time and reagents
Solution Approach 1:
The invention converts the potential harm of adaptor dimers into a benefit by using the presence of unreacted adaptors as a useful feature - the modified adaptors specifically bind to target DNA through their designed sequences, and any unreacted adaptors can be easily distinguished and removed by simple methods, thus achieving high sequencing accuracy without time-consuming purification
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 enhances library yield and reduces adaptor dimer production, improving sequencing efficiency and library preparation from as little as 24 pg of single-stranded DNA.
Implementation Method 1
TACS ligation is a highly efficient single-stranded DNA ligation technique and is a unique reaction that ligates single-stranded DNAs to each other, which was developed based on the fact that the reaction efficiency is particularly high when the 3'-end of nucleotides on which the RNA ligase acts is RNA
Implementation Method 2
a step of annealing a single-stranded adaptor sequence A2 to the adaptor sequence A1 portion contained in the adaptor-tagged single-stranded DNA and then elongating the 3'-end of the adaptor sequence A2 by DNA polymerase to produce adaptor-tagged double-stranded DNA
Implementation Method 3
a step of reacting topoisomerase with a double-stranded adaptor sequence B1, which has only one 5'-end phosphorylated and has a topoisomerase recognition sequence in the region containing the 3'-end complementary to said 5'-end, to produce a topoisomerase-linked double-stranded adaptor sequence B2 having topoisomerase conjugated to the 3'-end of said recognition sequence
Implementation Method 4
a step of performing PCR using the double-stranded DNA having both ends tagged with adaptor sequences as a template to obtain a library containing a plurality of said double-stranded DNAs
Data Source
Figure 1A~1D
Figure 2A~2B
Figure 3A~3C
AI summary
The present invention relates to a method for preparing a double-stranded DNA library from single-stranded DNA fragments and provides a highly efficient library preparation method, etc., that generates or contains few adapter dimers. The present invention is a method for preparing a double-stranded DNA library from single-stranded DNA fragments that includes the specific steps (i) to (v).