Multiplex Nucleic Acid Analysis Using Universal Primers
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Solution Overview
Problem
Current nucleic acid analysis techniques are either not easily multiplexed, time-consuming, expensive, or inaccurate, limiting their efficiency in detecting genetic aberrations and other genetic information.
Innovation Solution
A method involving the use of probes and primers with specific binding sites and stuffer sequences, combined with ligation and amplification reactions, allows for multiplexed nucleic acid analysis, enabling the simultaneous detection of multiple target nucleic acids in a sample through capillary electrophoresis.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If traditional nucleic acid analysis techniques (OLA, LCR, microarrays, high throughput sequencing) are used, then detection capability is achieved, but the methods are either not easily multiplexed, time-consuming, expensive, or inaccurate
Solution Approach 1:
The patent employs universal primer binding sites incorporated into probe structures that can be simultaneously amplified by a single set of primers through PCR. This multi-functional design allows multiple different target nucleic acids to be detected in a single multiplexed reaction, resolving the contradiction between versatility and productivity by enabling one amplification system to handle multiple targets efficiently
Solution Approach 2:
The patent divides the detection system into modular probe components, each with specific binding regions and universal primer binding sites. This segmentation allows independent design and optimization of each probe for different targets while maintaining compatibility with a unified amplification and detection system, enabling easy multiplexing without sacrificing analysis efficiency
2Measurement precision
If traditional nucleic acid analysis techniques are used, then detection accuracy is achieved, but the methods are time-consuming and expensive
Solution Approach 1:
The patent combines hybridization, amplification, and detection steps into an integrated workflow where multiple probes are simultaneously amplified using a single PCR reaction with universal primers. This merging of operations maintains detection accuracy through specific probe-target binding while dramatically reducing analysis time and cost by eliminating the need for separate amplification reactions for each target
Solution Approach 2:
The patent uses PCR amplification to generate multiple copies of the probe-target hybrids, enabling sensitive detection of low-abundance targets. This copying mechanism maintains measurement precision by ensuring sufficient signal for accurate detection while reducing analysis time through exponential amplification rather than linear detection methods
3Productivity
If simple detection methods are used, then speed is improved, but accuracy and multiplexing capability deteriorate
Solution Approach 1:
The patent replaces complex mechanical separation and individual analysis of multiple targets with a molecular biology-based solution where universal primers selectively amplify multiple probe-target hybrids simultaneously. This substitution maintains high analysis speed while preserving detection accuracy through the specificity of nucleic acid hybridization and amplification
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 multiplex nucleic acid analysis, allowing for the detection of multiple target nucleic acids, including SNPs, CNVs, and other genetic aberrations, in a single assay, improving the speed and accuracy of genetic testing.
Implementation Method 1
hybridizing the set of probes to the complementary regions of target nucleic acids
Implementation Method 2
performing a ligation reaction on the hybridized probes to form a ligation product
Implementation Method 3
amplifying the ligation product with a set of primers to obtain an amplification product
Implementation Method 4
determining the presence, absence or quantity of the ligation product in the amplification product by capillary electrophoresis
Data Source
AI summary
Here provided is a method for multiplex nucleic acid analysis. The method includes steps of hybridizing sets of probes to target nucleic acids in a sample, ligating the hybridized probes, amplifying the ligated probes, and assaying the amplification products to determining the presence, absence, or quantity of the target nucleic acids in the sample. The multiplexity is made available in part by adding detectable moieties and inserting stuffer sequences in primers so that amplification products may be identified on the basis of the detectable moieties and fragment sizes. Also provided is a sensitive method of detecting small copy number changes by measuring the copy number of a plurality of target sites in the nucleic acid in a test sample in comparison to a control sample and then determining the copy number of the nucleic acid based on the measured copy number of the plurality of target sites. Further provided is a kit for multiplex nucleic acid analysis and for small copy number change determination.