Circularized Nucleic Acid Templates for Fewer Ligation Steps
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Solution Overview
Problem
Existing methods for preparing DNA libraries for sequencing, particularly for DNA methylation profiling, involve multiple ligation steps that reduce conversion efficiency and require additional cleanup steps, complicating the process.
Innovation Solution
A method involving the circularization of nucleic acid templates using linkers with primer extension reaction terminating organic molecules, such as polyalkylene glycol or phosphonamidite, to simplify the preparation and enhance efficiency by reducing ligation steps and improving library conversion efficiency.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If multiple ligation steps are used for adding sequencing adapters, then adapter ligation can be achieved, but conversion efficiency is reduced and process complexity increases
Solution Approach 1:
The patent combines multiple ligation steps into a single ligation reaction by using a bifunctional linker molecule that can ligate to both the 5′ and 3′ ends of the DNA fragment simultaneously. This merging of operations reduces the number of separate ligation reactions required, thereby improving conversion efficiency and simplifying the overall process while maintaining complete adapter attachment.
Solution Approach 2:
The adapter is divided into two separate functional components: a 5′ adapter portion and a 3′ adapter portion, which are connected through a central linker. This segmentation allows each portion to be optimized for its specific ligation site while the linker provides the necessary spacing and structural integrity, enabling efficient single-step ligation without the need for sequential adapter additions.
2Reliability
If multiple ligation steps are performed, then complete adapter attachment is achieved, but additional cleanup steps are required
Solution Approach 1:
By merging the attachment of both 5′ and 3′ adapters into a single ligation reaction step, the patent eliminates the need for intermediate cleanup steps that would otherwise be required between sequential ligation reactions. The bifunctional linker enables both adapters to be attached simultaneously, reducing total process time while ensuring complete adapter coverage.
3Ease of manufacture
If traditional adapter ligation method is used, then sequencing adapters are added, but library conversion efficiency is reduced
Solution Approach 1:
The patent introduces a bifunctional linker molecule as an intermediary that mediates the attachment of both 5′ and 3′ adapters to the DNA fragment in a single reaction. This intermediary component facilitates efficient adapter ligation by providing a structural bridge between the two adapter portions, thereby improving library conversion efficiency while maintaining the ease of adapter addition.
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 reduces the number of ligation steps and cleanup processes, leading to improved overall library conversion efficiency and simplification of DNA library preparation for sequencing.
Implementation Method 1
copying the target sequence by a primer extension reaction using a polymerase
Implementation Method 2
L is a linker including a primer extension reaction terminating organic molecule, e.g., the linker L joins PS1 and PS2 and includes an organic molecule that terminates polymerization in a primer extension reaction
Implementation Method 3
the structure is circularized by binding a 5′ end thereof to a 3′ end thereof
Data Source
AI summary
Compositions and methods are provided for amplifying nucleic acids, including cell free nucleic acid fragments, in preparation for sequencing. Methods are provided for making circularized nucleic acid templates having the structure [T]-[PS1]-[L]-[PS2] or [PS1]-[L]-[PS2]-[T′], where (a) T is a target nucleic acid and T′ is a complement to a target nucleic acid; (b) each of PS1 and PS2 is a nucleic acid primer site; (c) L is a linker having a primer extension reaction terminating organic molecule; and the structure is circularized by binding a 5′ end thereof to a 3′ end thereof. Target sequences in the circularized templates are amplified by binding to PS1 a primer complimentary to PS1 and binding to PS2 a primer complimentary to PS2 and copying the target sequences by a primer extension reaction. Advantages include a reduction in ligation steps, which can result in fewer clean up steps and improved library conversion efficiency.