Bubble Primer Asymmetric Adapter Integration
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Solution Overview
Problem
Current DNA sequencing methods, such as next-generation sequencing (NGS), face inefficiencies due to random adapter attachment, leading to a high proportion of non-amplifiable fragments and unnecessary sequencing of known regions, which increases costs and reduces fidelity of unknown sequence data.
Innovation Solution
A method using 'bubble primers' that amplify specific regions of interest with a second primer having complementary and non-complementary portions, allowing asymmetric integration of adapters and minimizing known sequence remnants, enabling targeted sequencing with reduced unnecessary sequencing.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If random adapter ligation is used to generate sequencing libraries, then all DNA fragments can be processed in parallel, but about half of the fragments become non-amplifiable due to symmetric adapter attachment (A/A or B/B)
Solution Approach 1:
The patent applies preliminary action by performing a first amplification step before adapter ligation to generate fragments with predetermined asymmetric ends. The first primer pair introduces initial sequences that create distinct 5' and 3' ends, ensuring that subsequent adapter ligation produces predominantly asymmetric A/B products rather than symmetric A/A or B/B products. This pre-preparation of asymmetric structures before the main sequencing library construction resolves the contradiction by ensuring amplifiability is established in advance.
Solution Approach 2:
The patent directly applies asymmetry by designing a two-step amplification process using different primer pairs (first and second primer pairs) that create fragments with inherently asymmetric end structures. The first amplification generates fragments with specific 5' and 3' ends, and the second amplification maintains this asymmetry. This deliberate creation of asymmetric fragment ends ensures that adapter ligation produces amplifiable A/B products rather than non-amplifiable symmetric products, resolving the reliability issue while maintaining high productivity.
2Quantity of substance
If standard PCR amplification is used to enrich target regions, then target sequences are amplified to outnumber non-target regions, but large primer remnant regions are inserted between adapters and target DNA
Solution Approach 1:
The patent applies the taking out principle by using a second primer pair in the second amplification step that is designed to be substantially identical to the first primer pair. This design choice ensures that the primer sequences are minimized and positioned optimally, extracting unnecessary remnant sequences from the final product. The primers are configured to bind directly adjacent to the target region of interest, thereby removing excessive known sequence remnants that would otherwise require unnecessary sequencing cycles and reduce the fidelity of unknown sequence data.
Solution Approach 2:
The patent applies parameter changes by modifying the primer design parameters - specifically making the second primer pair substantially identical to the first primer pair. This parameter change optimizes the amplification process to produce fragments with minimal primer remnants. By adjusting the primer sequence identity and binding position parameters, the patent reduces the length of known sequence regions that must be sequenced, thereby improving the efficiency and fidelity of unknown sequence determination.
3Ease of manufacture
If ligation-based adapter integration is used, then adapters can be attached to fragmented DNA, but asymmetric integration (different adapters on each end) is difficult to achieve
Solution Approach 1:
The patent applies mechanics substitution by replacing the mechanical ligation process with a biochemical amplification-based approach. Instead of using enzymatic ligation to attach adapters to fragmented DNA ends (which struggles to achieve asymmetric integration), the patent uses two sequential PCR amplification steps with specifically designed primer pairs. The first amplification creates fragments with defined asymmetric ends, and the second amplification maintains this asymmetry. This substitution of the mechanical ligation system with a biochemical amplification system achieves precise asymmetric integration inherently, without requiring complex control mechanisms.
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 generates sequencing-ready fragments with controlled adapter integration, reducing resource usage and improving the fidelity of unknown sequence data, making targeted sequencing more efficient and cost-effective.
Implementation Method 1
the second primer comprises a nucleic acid sequence having a first portion which is complementary to a first portion of the starting template, a second portion which is not complementary to the starting template, and a third portion which is complementary to a second portion of the starting template; wherein the first and second portions of the starting template are adjacent or in close proximity to one another; wherein the first, second, and third portions of the second primer are arranged in that order from 5' to 3', such that on hybridisation to the starting template the second portion of the primer remains unhybridised and forms a loop between the first and third portions
Implementation Method 2
amplifying the region of interest from the starting template using a first primer pair to form an amplicon incorporating the region of interest, b) amplifying the region of interest from the first amplicon generated in step a) using a nucleic acid amplification reaction with a second primer, to form an amplicon incorporating the second primer
Data Source
Figure 1
Figure 2a~2e
AI summary
A method for generating sequence ready fragments of nucleotide sequences is described, the method making use of "bubble primers" which include first and third portions which hybridise to a target, and a second partly self-complementary portion which forms an unhybridised loop. The loop contains generic sequences allowing use of sequencing primers. The first portion may be degradable so as to generate an amplicon of sequence of interest flanked by the third portion and the generic sequences of the second portion. In preferred embodiments, the second portion, or the region between the second portion and the third portion, also comprises a tetrad of nucleotides A, C, G, T, allowing calibration of the sequencing reaction.