Nucleic Acid Amplification Using Folded Adapter DNA
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Massively parallel nucleotide sequencing faces challenges in throughput and accuracy due to time-consuming and labor-intensive cluster disposition on flow path substrates, and conventional amplification methods produce unintended amplification products and vary in primer hybridization efficiency, affecting the quality of DNA libraries.
Innovation Solution
The method involves ligating adapters capable of forming folded structures to template DNA to create cyclic DNA templates, which are then amplified without primers, resulting in concatemers suitable for nucleotide sequence analysis, eliminating the need for primer binding and maintaining DNA abundance ratios.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If conventional PCR or RCA amplification methods are used with primers, then amplification of template DNA can be achieved, but unintended amplification products are generated and primer hybridization efficiency varies, affecting DNA library quality
Solution Approach 1:
The invention extracts and eliminates the primer component from the amplification system. By using a nick in the cyclic DNA template as the starting point for elongation rather than external primers, the method removes the source of unintended amplification products and hybridization efficiency variations that plagues conventional PCR and RCA methods
Solution Approach 2:
The cyclic DNA template with a nick structure serves its own function as the amplification starting point. The nick provides the 3'-OH group necessary for DNA polymerase to initiate elongation without requiring external primers, making the system self-sufficient and eliminating primer-related artifacts
2Productivity
If many clusters are disposed on a flow path substrate using conventional methods, then massively parallel sequencing can be performed, but it takes much time and effort to dispose clusters, becoming an obstacle to improving throughput
Solution Approach 1:
The invention performs preliminary formation of concatemers with folded structures that naturally facilitate high-density positioning. By pre-configuring the DNA molecules into compact, structured units before substrate deposition, the method eliminates time-consuming manual or mechanical cluster disposition steps while maintaining high sequencing throughput
3Quantity of substance
If primers are introduced in sufficient amounts for amplification, then many DNA fragments can be amplified, but partial and tentative binding of primers to each other results in unintended amplification products
Solution Approach 1:
The invention removes primers from the amplification mixture entirely. By using the nicked cyclic template itself as the initiation site, the method eliminates primer-primer interactions that lead to dimer formation and unintended amplification products, while still enabling amplification of numerous 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
This approach enables efficient and accurate nucleic acid amplification, reducing the generation of artifacts, saving time and effort, and producing optimally-shaped concatemers for high-throughput nucleotide sequencing.
Implementation Method 1
adapтеры, содержащие последовательности, способные формировать структуру с перегибом
Implementation Method 2
elongation reaction starting from the nick as an origin using a strand-displacement DNA polymerase
Implementation Method 3
elongation reaction starting from the nick as an origin
Data Source
Figure 1(a)~1(f)
Figure 2(a)~2(c)
Figure 3(a)~3(c)
AI summary
An object of the present invention is to provide a method and means for amplifying a nucleic acid that reduce the time and effort required for amplification of template DNA and address the problems of conventional amplification methods, as well as a method and means for determining a nucleotide sequence using the same. The nucleic acid amplification method comprises the steps of: ligating a double-stranded adapter (20) containing adapter DNA strands capable of forming a folded structure to a double-stranded DNA (1, 2) containing a target DNA sequence (1) to prepare a cyclic DNA template composed of double-stranded DNA containing a nick (5); and performing a 3'-end elongation reaction using a strand-displacement DNA polymerase from the nick (5) as an origin, thereby producing a concatemer (29) in which a plurality of the target DNA sequences (1) and the adapter DNA strands capable of forming the folded structure are linked in series as a single-stranded DNA, wherein the concatemer (29) contains a plurality of the target DNA sequences (1) suitable for nucleotide sequence analysis and has a folded shape such that it takes the form of a ball due to its folded structure.