Nucleic Acid Quantification via Random Sequence Tagging
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Solution Overview
Problem
Current methods for quantifying target nucleic acids face challenges due to heterogeneous amplification efficiency of PCR, which complicates accurate sequencing and quantification, especially when ligation reactions are required and restriction enzyme efficiency varies.
Innovation Solution
A method involving single primer extension and PCR using primers with specific and random sequences to generate double-stranded nucleic acids, allowing for sequencing and quantification without ligation, using the diversity of random sequences to determine target nucleic acid copy numbers through next-generation sequencing.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Ease of manufacture
If a specific sequence is ligated to the terminal of a target nucleic acid using restriction enzymes, then the target nucleic acid can be prepared for sequencing, but the formation of protruding ends may be difficult or impossible depending on the target sequence, and restriction enzyme cleavage efficiency varies
Solution Approach 1:
The invention extracts and removes the problematic ligation step requiring restriction enzymes. Instead of ligating adapters to target sequences, the method directly synthesizes double-stranded nucleic acids containing the target sequence through primer extension, eliminating the unreliable restriction enzyme cleavage and ligation steps while maintaining the ability to prepare sequencing templates.
Solution Approach 2:
The invention introduces a mediator approach by using primers containing random sequences as intermediaries. These primers serve as bridges that anneal to the target sequence and enable synthesis of double-stranded nucleic acids without requiring restriction enzyme cutting or adapter ligation, thus resolving the contradiction between ease of preparation and reliability.
2Quantity of substance
If PCR amplification is performed to increase target sequence abundance for detection, then the target sequence becomes detectable, but amplification efficiency varies depending on sequence, GC content and secondary structure, compromising quantitative accuracy
Solution Approach 1:
The invention applies preliminary action by performing a primer extension reaction before PCR amplification. This initial synthesis step creates double-stranded nucleic acids with incorporated random sequences that serve as unique molecular identifiers. By establishing this structure beforehand, the subsequent PCR amplification preserves the original target sequence ratios while generating sufficient abundance for detection, thus maintaining both quantity and measurement precision.
Solution Approach 2:
The invention uses copying by incorporating random sequences during the primer extension step, creating multiple copies of the target sequence each tagged with a unique random sequence identifier. This allows the amplified copies to be traced back to their original molecules, enabling accurate quantification even after extensive PCR amplification, thus resolving the contradiction between abundance and quantitative accuracy.
3Productivity
If relative abundance in sequence library is used to estimate target sequence copy number, then sequencing can be performed, but it is difficult to accurately estimate copy number due to heterogeneous amplification efficiency
Solution Approach 1:
The invention uses copying with unique molecular identifiers (random sequences) incorporated during primer extension. Each original target molecule is copied multiple times during PCR, but each copy carries the original random sequence tag. By counting the diversity of random sequences in the sequenced library, the original copy numbers can be accurately reconstructed, maintaining both high sequencing productivity and precise copy number estimation.
Solution Approach 2:
The invention implements feedback by using the random sequence diversity as a molecular barcode system. The sequencing data provides feedback about the original sample composition through the distribution of random sequences, allowing accurate reconstruction of initial copy numbers despite heterogeneous amplification efficiency during PCR, thus resolving the contradiction between productivity and measurement precision.
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 accurate and simultaneous sequencing and quantification of target nucleic acids, overcoming the limitations of conventional methods by using the diversity of random sequences to estimate copy numbers with high precision, applicable to both mock and environmental samples.
Implementation Method 1
carrying out an annealing reaction, a nucleic acid extension reaction, and a nuclease reaction by using as a template a single-stranded nucleic acid of target nucleic acid or a single-stranded nucleic acid obtained from a double-stranded nucleic acid of target nucleic acid
Implementation Method 2
carrying out an annealing reaction, a nucleic acid extension reaction, and a nuclease reaction by using as a template a single-stranded nucleic acid of target nucleic acid
Implementation Method 3
carrying out an annealing reaction, a nucleic acid extension reaction, and a nuclease reaction by using as a template a single-stranded nucleic acid of target nucleic acid
Implementation Method 4
carrying out a PCR using the first double-stranded nucleic acid obtained from the step (1) as a template along with a second primer containing the second sequence and a third primer containing the specific sequence so as to obtain a second double-stranded nucleic acid
Data Source
Figure 1(1)~1(6)
Figure 2
Figure 3
AI summary
The purpose of the present invention is to provide a method for allowing both sequencing and quantification of a target nucleic acid without performing a ligation reaction and while solving a problem which arises due to PCR amplification efficiency. The method comprises (1) performing a single primer extension reaction using a primer having a sequence specific to the sequence of a target nucleic acid, and a random sequence and a known sequence which are introduced upstream of that specific sequence, thereby obtaining a double-stranded nucleic acid having the random sequence and the sequence of the target nucleic acid, (2) performing PCR using the obtained double-stranded nucleic acid as a template, and a primer specific to a known sequence and a primer having a portion of the sequence of the target nucleic acid, thereby obtaining an amplified product of the double-stranded nucleic acid having the random sequence and the sequence of the target nucleic acid, and (3) performing sequencing using the obtained amplified product of the double-stranded nucleic acid, thereby decoding the sequence of the random sequence as well as the target nucleic acid.