SNP 16S DNA Internal Standard Corrects PCR Amplification Bias
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Solution Overview
Problem
Gene-based microbial community analysis methods, particularly those using 16S rDNA gene amplification, face challenges in quantitative accuracy due to biases in amplification efficiency across different microorganisms, which are exacerbated by next-generation sequencing (NGS) and PCR processes.
Innovation Solution
A method involving the preparation of single nucleotide polymorphism (SNP) 16S DNA, where the nucleotide sequence is partially substituted to introduce SNPs at non-primer binding sites, allowing for the use of this SNP 16S DNA as an internal standard to adjust amplification efficiency by quantifying both SNP and 16S rDNA through nucleotide sequencing.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If 16S rDNA gene amplification is used for microbial community analysis, then various microorganisms can be simultaneously detected with high sensitivity and precision, but amplification bias occurs depending on the microorganism due to differences in genome extraction and amplification efficiency
Solution Approach 1:
The patent introduces an intermediary substance (SNP 16S DNA with artificial single nucleotide polymorphisms) that serves as a reference standard to mediate between the PCR amplification process and the quantification step. This intermediary allows for the calculation of amplification efficiency ratios, thereby correcting the quantitative bias introduced by differential amplification of different microorganisms while preserving the high sensitivity and precision of the original 16S rDNA gene amplification method
2Adaptability or versatility
If degenerate primers are used to amplify 16S rDNA genes from multiple microorganisms, then broad coverage of different microorganism genes is achieved, but amplification bias increases depending on the microorganism
Solution Approach 1:
The patent applies parameter changes by introducing artificial single nucleotide polymorphisms (SNPs) at specific positions in the 16S rDNA gene sequence. By modifying the nucleotide sequence parameters at non-primer binding sites, the patent creates distinguishable reference standards that allow for the calculation and correction of amplification efficiency variations, thereby maintaining broad microorganism coverage while improving amplification consistency through quantitative normalization
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 improves the accuracy of microbial community analysis by normalizing amplification efficiency, providing precise quantification of microorganisms and their effects, such as antibiotic resistance, across various environments and samples.
Implementation Method 1
preparing a single nucleotide polymorphism (SNP) 16S DNA of which a nucleotide sequence is partially substituted so as to have a single nucleotide polymorphism (SNP)
Data Source
AI summary
The present invention relates to utilization of an artificially synthesized nucleic acid, and more particularly, to a quantitative analysis method capable of quantitatively adjusting gene-based microbial community analysis results by preparing a microorganism 16S rDNA gene, which has a single nucleotide polymorphism (SNP) at a particular location so as to be differentiated from a gene of a target microorganism on the nucleotide sequence, and then using the microorganism 16S rDNA gene as an internal standard material which is quantifiable through nucleotide sequencing.