Multiplex PCR for Fetal Aneuploidy Diagnosis
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Solution Overview
Problem
Current non-invasive prenatal diagnostic methods for fetal chromosomal aneuploidy, such as massive parallel sequencing, are expensive, have low throughput, and suffer from quantitative bias, particularly for chromosomes 18 and 13.
Innovation Solution
A multiplex-PCR based approach that amplifies selected chromosomal regions from both suspected aneuploid and euploid chromosomes in a single quantitative reaction, followed by DNA sequencing and normalization to determine dosage quotients indicative of aneuploidy.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If massive parallel sequencing is used for non-invasive prenatal diagnosis, then comprehensive chromosomal analysis is achieved, but the cost increases and throughput decreases
Solution Approach 1:
The patent segments the genome into specific chromosomal regions of interest (chromosomes 13, 18, 21, X, and Y) and designs targeted PCR assays for these regions. This segmentation allows the method to focus sequencing resources on clinically relevant areas rather than performing whole-genome sequencing, thereby reducing cost and increasing throughput while maintaining detection accuracy for aneuploidies.
Solution Approach 2:
The patent changes the sequencing depth parameter from high coverage required for whole-genome analysis to optimized lower coverage (10-100x) sufficient for targeted regional analysis. This parameter change, combined with the targeted approach, reduces the overall sequencing cost and increases throughput while maintaining the reliability needed for detecting chromosomal imbalances.
2Reliability
If massive parallel sequencing is used for non-invasive prenatal diagnosis, then comprehensive chromosomal analysis is achieved, but the cost increases
Solution Approach 1:
The patent segments the genome into specific chromosomal regions of interest (chromosomes 13, 18, 21, X, and Y) and designs targeted PCR assays for these regions. This segmentation allows the method to focus sequencing resources on clinically relevant areas rather than performing whole-genome sequencing, thereby reducing cost and increasing throughput while maintaining detection accuracy for aneuploidies.
Solution Approach 2:
The patent employs disposable PCR reaction mixes and sequencing libraries that are optimized for targeted regions. By using pre-designed primers and probes for specific chromosomal regions rather than universal whole-genome reagents, the method reduces per-sample cost while maintaining diagnostic reliability.
3Measurement precision
If quantitative PCR is used to amplify chromosomal regions, then sensitivity and specificity improve, but the risk of amplification bias increases
Solution Approach 1:
The patent designs multiple PCR assays targeting different regions within each chromosome of interest, ensuring that each assay is optimized for its specific local genomic context. This local optimization minimizes amplification bias by selecting regions with similar GC content and complexity, thereby maintaining both sensitivity and quantitative accuracy.
Solution Approach 2:
The patent optimizes PCR reaction parameters including cycle number, primer concentration, and annealing temperature to achieve quantitative amplification. By carefully controlling these parameters and using a limited number of cycles (typically 10-20), the method minimizes amplification bias while maintaining sufficient sensitivity for detecting chromosomal imbalances.
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
Achieves high sensitivity and specificity, approaching 100% for the simultaneous detection of chromosome 13, 18, 21, X, and Y aneuploidies, while reducing costs and improving throughput compared to existing methods.
Implementation Method 1
amplifying a selected set of target DNA sequences from one or more chromosomes which are suspected to be aneuploid in the fetus... and amplifying a selected set of target DNA sequences from one or more reference chromosomes
Implementation Method 2
conducting DNA sequencing of said amplified selected set of target DNA sequences to determine the sequence of said DNA sequences
Data Source
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AI summary
The invention relates to prenatal detection methods using non-invasive techniques. In particular, it relates to prenatal diagnosis of a fetal chromosomal aneuploidy by detecting fetal and maternal nucleic acids in a maternal biological sample. More particularly, the invention applies multiplex PCR to amplify selected fractions of the respective chromosomes of maternal and fetal chromosomes. Respective amounts of suspected aneuploid chromosomal regions and reference chromosomes are determined from massive sequencing analysis followed by a statistical analysis to detect a particular aneuploidy.