Allele-Specific Probe Arrays for Mixed DNA Copy Number Detection
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Solution Overview
Problem
Current microarray technologies struggle to accurately differentiate between highly similar nucleic acid subpopulations in mixed samples, such as fetal and maternal DNA in maternal blood or tumor and normal cells, leading to high false-positive rates and the need for invasive diagnostic procedures.
Innovation Solution
The method involves using molecular inversion probes to selectively enrich and genotype polymorphic sites, allowing for the detection of copy number variations and genotyping in mixed nucleic acid populations by analyzing signals from both major and minor subpopulations on a single array.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If conventional microarray analysis is used on mixed nucleic acid samples, then the analysis is cheaper and quicker than sequencing, but it cannot reliably differentiate between highly similar subpopulations (e.g., fetal vs. maternal DNA, tumor vs. normal cells)
Solution Approach 1:
The method segments the mixed nucleic acid population into distinct subpopulations by targeting polymorphic sites. Allele-specific probes are designed to bind to specific alleles at polymorphic loci, enabling separate detection and quantification of fetal and maternal DNA, or tumor and normal cells, based on their unique genetic markers.
Solution Approach 2:
The invention applies local quality by focusing analysis on specific polymorphic sites rather than attempting to analyze the entire genome. By selecting informative polymorphic markers that differ between subpopulations, the method achieves high differentiation precision at targeted locations while maintaining overall cost-effectiveness.
2Device complexity
If single or low multiplex assays are used, then the assay complexity is reduced, but the ability to detect low concentration subpopulations (e.g., fetal DNA at 4-15% of total cell-free DNA) is insufficient
Solution Approach 1:
The method employs a universal genotyping assay platform that can simultaneously detect multiple polymorphic sites in a single reaction. This multi-plex approach allows concurrent analysis of numerous genetic markers, enhancing the ability to detect low-abundance subpopulations through statistical analysis of multiple loci while maintaining manageable assay complexity.
Solution Approach 2:
The invention uses copy number analysis of polymorphic sites to detect subpopulations. By analyzing the relative copy numbers of different alleles across multiple polymorphic loci, the method can statistically infer the presence and proportion of minor subpopulations even when they constitute only 4-15% of the total nucleic acid sample.
3Ease of operation
If non-invasive sampling methods are used, then sample collection is easier and less risky, but the limited amount of nucleic acid available (5-15 ng from 10 mls blood) complicates the analysis
Solution Approach 1:
The method extracts and enriches specific polymorphic sites from the limited nucleic acid sample using allele-specific probes. By focusing the analysis on targeted polymorphic regions rather than attempting to analyze the entire genome, the invention maximizes the information obtained from the small amount of available nucleic acid (5-15 ng).
Solution Approach 2:
The invention changes the analytical parameters by shifting from whole-genome analysis to targeted polymorphic site analysis. This parameter change allows efficient use of limited nucleic acid material while achieving sufficient statistical power to detect subpopulations through analysis of multiple polymorphic markers.
4Ease of operation
If conventional prenatal testing (maternal serum screening and ultrasound) is used, then the testing is non-invasive, but the sensitivity and specificity are very poor leading to high false positive rates
Solution Approach 1:
The invention replaces mechanical/physical screening methods (ultrasound) and biochemical screening (maternal serum markers) with molecular genetic analysis. By directly analyzing the DNA sequence and polymorphic markers of cell-free fetal DNA in maternal blood, the method achieves high sensitivity and specificity while remaining non-invasive, eliminating the need for invasive diagnostic procedures.
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 enhances the sensitivity and specificity of microarray analysis, reducing false-positive rates and enabling accurate detection of genetic abnormalities in non-invasive samples, such as fetal aneuploidy and tumor genetics, without the need for invasive procedures.
Implementation Method 1
hybridizing at least one nucleic acid fragment containing or derived from the nucleic acid population and containing the polymorphic site to an oligonucleotide probe of an oligonucleotide array
Data Source
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AI summary
This disclosure provides methods and systems useful in array-based analysis of mixed nucleic acid populations, including for multiplex genotyping of a mixed nucleic acid sample and for detecting differences in copy number of a target polynucleotide and/or a target chromosome (e.g., microdeletions, duplications and aneuploidies). The disclosure also provides methods and systems useful in the diagnosis of genetic abnormalities in a mixed nucleic acid population taken non-invasively from an organism, such as a sample of blood, plasma, serum, urine stool or saliva. The disclosed methods and systems find use in multiple applications, including prenatal testing and cancer diagnostics. The disclosure is based on the hybridisation of amplified fragments from the sample, e.g. a maternal sample, which may employ molecular inversion probes MIP to an oligonucleotidfe array and the detection of the alleles based on different signals from the different alleles of the SNP. The disclosure also discloses how the determination of the allele ratio may be used in the determination of fetal and maternal CNVs, e.g. aneuploidies.