DNA Adapter Molecules with Terminal Modifications for NGS Library Preparation
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Solution Overview
Problem
Current methods for preparing DNA libraries for Next Generation Sequencing require additional steps to modify DNA ends before adapter ligation, which can lead to adapter dimer and oligomer formation, and necessitate inactivating enzymes, making the process complex and error-prone.
Innovation Solution
DNA-adapter-molecules with specific terminal modifications that prevent binding with kinases and polymerases, allowing for blunt-end ligation without enzyme inactivation, enabling direct addition to fragmented DNA during library preparation and reducing dimer and oligomer formation.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If common adapter sequences are added to the end repair mix, then the adapter ligation can be performed, but the enzymes in the end repair mix modify the adapters causing loss of directionality and formation of adapter dimers and oligomers
Solution Approach 1:
The adapter sequences are designed with asymmetric modifications: the 5' end of the first strand and 5' end of the reverse strand are modified to prevent kinase binding, while the 3' end of the reverse strand retains a free hydroxyl group for ligation. This local differentiation of chemical properties at different ends of the adapter enables selective enzymatic reactions and prevents unwanted modifications, resolving the contradiction between ligation efficiency and adapter purity.
Solution Approach 2:
The adapter sequences are pre-modified with 5' end modifications before being added to the end repair mix. This preliminary modification prevents kinases and polymerases in the end repair mix from acting on the adapters, thereby preventing the formation of adapter dimers and oligomers while maintaining the free 3' hydroxyl group needed for subsequent ligation to DNA fragments.
2Reliability
If additional enzyme inactivation steps are introduced to prevent adapter modification, then adapter purity is maintained, but the process complexity and error-proneness increase
Solution Approach 1:
The protective 5' end modifications are incorporated into the adapter sequences during their synthesis, before the library preparation process begins. This preliminary action ensures that when adapters are added to the end repair mix, they are already protected against unwanted enzymatic modifications, eliminating the need for additional enzyme inactivation steps and simplifying the overall workflow.
Solution Approach 2:
The adapter sequences are designed to be self-protecting through their modified 5' ends. The modifications inherently prevent kinase and polymerase binding without requiring external protection mechanisms or additional processing steps. The adapters essentially protect themselves from unwanted modifications, reducing process complexity while maintaining purity.
3Manufacturing precision
If unphosphorylated DNA adapters are used for ligation, then directionality is maintained, but phosphodiester linkages are not formed at the 5' end requiring additional nick translation steps
Solution Approach 1:
The adapter design implements local quality differentiation: the 5' ends are modified to prevent phosphorylation and maintain directionality, while the 3' end of the reverse strand retains a free hydroxyl group that can participate in ligation. This localized functional differentiation enables the adapter to maintain directionality while still forming stable ligations to the DNA fragments.
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
Simplifies the library preparation process by allowing adapters to be added early, minimizing enzyme inactivation steps and reducing errors, while maintaining efficient ligation and sequencing performance.
Implementation Method 1
the 5' end of the first strand is modified in a way, that the first nucleotide does not contain a free hydroxyl group and not a free phosphate at the 5' position, so that no binding site for kinases is available
Implementation Method 2
the 3' end of the first strand is modified in a way that the last nucleotide does not contain a free hydroxyl group at the 3' position, so that no ligation can occur
Implementation Method 3
the 3' end of the reverse strand features a free hydroxyl group (at the 3' position of the last nucleotide)
Implementation Method 4
First strand and reverse strand are annealed to each other by complementary base pairing, without any overhang (blunt ends)
Data Source
Figure 1~4
Figure 5(A)~5(B)
Figure 6(A)~6(B)
AI summary
The invention relates to DNA-adapter-molecules for the preparation of DNA-libraries and methods for producing them and their use. The invention is useful for the application in molecular biology, in particular for Next Generation Sequencing and/or Library Multiplexing. The present invention discloses DNA-adapter-molecules, comprising a double-stranded polynucleotide molecule, whereat the 5' end of the first strand is modified in a way, that no binding site for kinases is available, the 3' end of the first strand is modified in a way that no ligation can occur, the 5' end of the reverse strand is modified in a way, that no binding site for a kinase is available, and the 3' end of the reverse strand features a free hydroxyl group (at the 3' position of the last nucleotide).