Stopper-Guided Nucleic Acid Amplification for Uniform Coverage
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Existing methods for nucleic acid amplification and sequencing suffer from sequence bias and uneven coverage of sequence fragments, particularly at the 5' and 3' ends of the template, leading to reduced confidence in the generated joined sequence.
Innovation Solution
A method involving the use of nucleic acid stoppers to control elongation, followed by ligation of adaptor nucleic acids with unique identification sequences at the 5' end of elongation products, ensuring these sequences do not hybridize to the template or stoppers, thereby enhancing coverage and reducing sequence bias.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If standard nucleic acid amplification methods are used, then amplification efficiency is achieved, but sequence bias and uneven coverage occur particularly at the 5' and 3' ends of the template
Solution Approach 1:
The amplification process is segmented into controlled elongation steps using multiple primers with different extension lengths. Each primer generates fragments of specific lengths, creating a segmented coverage pattern that ensures uniform representation across the entire template including 5' and 3' ends. This segmentation prevents any single region from being over- or under-represented in the final amplification product.
Solution Approach 2:
Different primers are designed with specific local properties - varying extension lengths and binding positions - to address local coverage needs. Primers are strategically positioned to ensure adequate coverage of regions that typically suffer from bias (5' and 3' ends), while maintaining appropriate coverage of central regions. This local optimization of primer properties achieves uniform sequence coverage throughout the template.
2Reliability
If multiple primers are used for amplification, then coverage is increased, but assembly and allocation of fragments to joined sequence becomes more difficult
Solution Approach 1:
Adaptor sequences serve as intermediary elements that facilitate fragment assembly. These known sequences are ligated to the 3' ends of amplified fragments and act as mediators during the assembly process. The adaptors provide standardized interfaces that simplify the allocation and joining of fragments to reconstruct the original template sequence, reducing the complexity of assembling multiple primer-generated fragments.
3Quantity of substance
If PCR amplification is performed to increase template amount, then sensitivity is improved, but sequence-specific PCR biases are introduced
Solution Approach 1:
Unique molecular identifiers (UMIs) are attached to template molecules before PCR amplification occurs. This preliminary tagging allows subsequent bioinformatic identification and grouping of PCR duplicates. By performing this action before amplification, the method preserves the ability to distinguish original template molecules from their PCR copies, enabling correction of amplification biases in the analysis phase while still achieving the necessary template quantity increase.
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 improves sequence coverage at both the 5' and 3' ends of the template, allowing for more accurate assembly and analysis of nucleic acid sequences by reducing sequence-dependent amplification bias and increasing the confidence in the generated joined sequence.
Implementation Method 1
annealing at primers to said template nucleic acid
Implementation Method 2
elongating the at least one oligonucleotide primer in a template specific manner thereby creating an elongation product
Implementation Method 3
ligating the adaptor nucleic acid at its 5' end to the 3' end of the elongation product
Data Source
Figure 1a~2a
Figure 2b~2c
Figure 3a~3e
AI summary
The present invention provides a method for generating labelled amplification fragments of a nucleic acid template comprising the steps of providing said template nucleic acid, annealing at least one oligonucleotide primer to said template nucleic acid, elongating the at least one oligonucleotide primer in a template specific manner thereby creating an elongation product, wherein said elongating reaction stops when the elongation product reaches the 5' end of the template nucleic acid or a nucleic acid elongation stopper that is annealed to the template nucleic acid downstream of the elongation product, providing an adaptor nucleic acid that comprises an identification sequence on its 5' end,wherein said identification sequence does not hybridize to the elongation stopper when in con- tact thereto, ligating the adaptor nucleic acid at its 5' end to the 3' end of the elongation product, thereby generating a labelled amplification fragment.