MLPA Multiplex Nucleic Acid Detection via Sequencing
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Solution Overview
Problem
Current Multiplex Ligation-dependent Probe Amplification (MLPA) techniques are limited to detecting about 40 target nucleic acid sequences in a single run due to the reliance on capillary electrophoresis-based detection systems, which restricts the number of queryable sequences and requires stuffer sequences for differentiation, increasing costs and complexity.
Innovation Solution
The method employs sequencing-by-synthesis (SBS) systems for detecting amplified products, allowing for the simultaneous querying of over 100 target nucleic acids without stuffer sequences, using adapter sequences and universal primers for amplification, and indexing for sample identification, independent of amplicon length.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Quantity of substance
If capillary electrophoresis-based detection systems are used, then the detection method is simple and established, but the number of queryable target nucleic acid sequences is limited to about 40 per run
Solution Approach 1:
The patent replaces the mechanical capillary electrophoresis separation system with a sequencing-by-synthesis detection system that uses fluorescently labeled nucleotides and imaging to detect amplicons. This substitution enables multiplexing of over 100 targets by using sequence information rather than size-based separation, thereby increasing the number of queryable targets while managing system complexity through different detection physics.
2Measurement precision
If stuffer sequences are included in probe sets for differentiation, then the detection accuracy is maintained, but the cost and probe set design complexity increase
Solution Approach 1:
The patent changes the differentiation parameter from size-based (using stuffer sequences in capillary electrophoresis) to sequence-based (using adapter sequences in sequencing-by-synthesis). This parameter change eliminates the need for stuffer sequences while maintaining detection accuracy, as the sequencing system can distinguish targets through their unique adapter sequences rather than requiring length variations.
Solution Approach 2:
The patent extracts and removes the stuffer sequence component from the probe set design. By using sequencing-by-synthesis detection, the system no longer requires stuffer sequences for target differentiation, thereby simplifying probe set design and reducing costs while maintaining the ability to accurately distinguish between different target nucleic acid sequences through adapter sequence identification.
3Reliability
If stuffer sequences are used for differentiation, then target identification is enabled, but the overall assay cost increases
Solution Approach 1:
The patent implements universal adapter sequences that can be used across all probe sets in the multiplex assay. These universal adapters enable target identification through sequencing without requiring individual stuffer sequences for each target, thereby reducing probe set design complexity and cost while maintaining reliable target identification capability through the universal sequencing approach.
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 significantly increases the number of queryable nucleic acid sequences per run, reduces costs by simplifying probe set design, enhances detection sensitivity, and facilitates simultaneous analysis of multiple samples with reduced sample size, while providing accurate copy number variation analysis.
Implementation Method 1
hybridization of the probe sets to their target nucleic acid sequences
Implementation Method 2
the adjacent half-probes are ligated
Implementation Method 3
the ligated probe sets are subjected to PCR amplification
Implementation Method 4
The amplified ligated probe sets are then analyzed by capillary electrophoresis (CE)
Implementation Method 5
detecting the at least 100 different amplicons in a detection system, independently of the length, by sequencing each of the at least 100 different amplicons
Data Source
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AI summary
Methods for multiplex ligation-dependent probe amplification include (a) providing sample tissue containing different target nucleic acids, (b) providing different probe sets for each of the target nucleic acids, each probe set including a first locus specific probe having a first adapter sequence and a first target specific portion and a second locus specific probe having a second adapter sequence, and a second target specific portion adjacent to the first target specific portion, (c) hybridizing the probe sets to the target sequences to form hybridization complexes, (d) ligating the hybridization complexes to form ligated probes, (e) amplifying the ligated probes to form amplicons, and (f) detecting the amplicons in a detection system by sequencing each of the amplicons.