Nucleotide Sequence Identification via Dual Primer Mobility
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Solution Overview
Problem
Current methods for nucleotide sequence identification of double-stranded DNA are labor-intensive and costly due to the complexity of preparing molecules with hairpin loops or cyclic structures, and often require sequencing both strands separately.
Innovation Solution
A method using two primers with target recognition sites and a reaction stop site to sequence both strands in a single reaction system, where the second primer's product has reduced mobility, allowing for distinct detection and analysis of both strands in one reaction.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If hairpin loop or cyclic molecule is used to link ends of double-stranded DNA, then both strands can be sequenced, but the work of linking and purification becomes complicated
Solution Approach 1:
The invention extracts only the necessary function of strand differentiation without requiring complex cyclic structures. By using two separate primers with distinct 5' ends, the patent achieves strand-specific sequencing through a simple linear DNA molecule, eliminating the need for hairpin loop formation and purification steps.
Solution Approach 2:
The patent segments the sequencing process into two independent primer-based reactions that can be performed simultaneously on the same DNA template. Each primer targets a specific strand, allowing both strands to be sequenced through a single linear DNA molecule without requiring cyclic linkage.
2Reliability
If both strands of double-stranded DNA are sequenced separately, then accuracy increases, but labor and cost increase
Solution Approach 1:
The invention merges the sequencing of both strands into a single reaction system by using two primers that can simultaneously bind to and sequence both strands of the DNA template. This combining approach maintains high accuracy through bidirectional sequencing while significantly improving efficiency by reducing the number of separate reactions required.
Solution Approach 2:
The patent creates a universal sequencing system where a single reaction mixture containing both primers can sequence both strands of the DNA template. This multi-functional approach allows one reaction system to perform the work of two separate sequencing reactions, thereby improving productivity without sacrificing accuracy.
3Productivity
If two primers are used to sequence both strands, then sequencing can be done in one reaction system, but the primers must be designed with specific features like reaction stop sites
Solution Approach 1:
The patent applies local quality by giving each primer specific localized features at its 5' end - one primer has a reaction stop site that prevents extension beyond a certain point, while the other primer has different characteristics. This localized differentiation allows the primers to be easily distinguished and their products to be separately analyzed without requiring complex overall primer designs.
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 reduces labor and cost by enabling efficient analysis of double-stranded DNA in a single reaction system, improving sequencing accuracy and efficiency by distinguishing between the two strands through electrophoresis.
Implementation Method 1
a step of identifying, by using a first primer having a target recognition site configured to form a complementary pair by hydrogen bonding with a part of one target polynucleotide of the target polynucleotide complementary pair
Implementation Method 2
a step of identifying, by using a second primer having a target recognition site configured to form a complementary pair by hydrogen bonding with a part of the other target polynucleotide of the target polynucleotide complementary pair
Implementation Method 3
a mobility of a second reaction product obtained by the second primer extending a complementary strand on the other target polynucleotide
Implementation Method 4
distinguishing between the two strands through electrophoresis
Data Source
AI summary
Provided is a nucleotide sequence identification method for analyzing a double-stranded DNA in one reaction system while reducing cost and labor. A method for identifying a nucleotide sequence in a target polynucleotide complementary pair constituting a double strand includes: a step of identifying, by using a first primer having a target recognition site configured to form a complementary pair with a part of one target polynucleotide of the target polynucleotide complementary pair, a nucleotide sequence in the one target polynucleotide; and a step of identifying, by using a second primer having a target recognition site configured to form a complementary pair with a part of the other target polynucleotide of the target polynucleotide complementary pair and a reaction stop site configured to stop a nucleotide extension reaction, a nucleotide sequence in the other target polynucleotide, in which a mobility of a second reaction product obtained by the second primer performing extension is smaller than a mobility of a first reaction product obtained by the first primer performing extension.