Barcode Positive Controls for Accurate Microdeletion PCR Detection
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Solution Overview
Problem
Subchromosomal abnormalities such as microdeletions and duplications are difficult to detect due to their small size, posing a challenge in early identification and management, particularly in prenatal testing, where they are as common as fetal autosomal trisomies and require improved detection methods.
Innovation Solution
The use of engineered nucleic acid constructs as positive controls, comprising a 5′ end region, central barcode region, and 3′ end region, specifically designed to replace microdeletion sequences, allowing for targeted amplification and accurate detection of microdeletions like 22q11.2, 5p15.2, 1p36, and 15q11-q13 deletions, with SNPs in the end regions for primer recognition.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If conventional detection methods are used for microdeletions, then the detection process is simple, but the detection accuracy is poor due to the small size of microdeletions
Solution Approach 1:
The patent creates engineered nucleic acid constructs that copy the structure of microdeletion regions, including flanking sequences and barcodes. These constructs serve as positive controls that replicate the target microdeletion architecture, enabling accurate detection through PCR amplification while maintaining simplicity in the overall detection workflow
Solution Approach 2:
The engineered constructs act as intermediaries between the detection system and the actual microdeletion targets. By incorporating barcodes and flanking sequences that mimic the microdeletion structure, these constructs enable the detection system to accurately identify microdeletions without requiring direct observation of the small abnormality itself
2Measurement precision
If engineered nucleic acid constructs with barcodes are used as positive controls, then the detection accuracy improves, but the construct design and preparation complexity increases
Solution Approach 1:
The engineered construct is segmented into distinct functional regions: 5' flanking sequence, central barcode region, and 3' flanking sequence. This segmentation allows each component to be designed and synthesized independently, simplifying the overall construction process while maintaining the functional integrity needed for accurate microdeletion detection
Solution Approach 2:
The patent utilizes parameter changes in nucleic acid sequence composition, specifically incorporating barcode sequences with defined lengths (6-9 base pairs) and specific GC content. These parameter optimizations enable reliable PCR amplification and detection while maintaining construct stability and specificity
3Productivity
If multiple microdeletion types are detected in a single assay, then the productivity increases, but the assay complexity and potential for false positives increases
Solution Approach 1:
The engineered construct design incorporates universal flanking sequences and barcode regions that can be detected across multiple microdeletion types. By using conserved sequencing primer binding sites and standardized barcode formats, the same assay platform can detect various microdeletions (e.g., 22q11.2, 5p15.2, 1p36, 15q11-q13) while maintaining reliable differentiation through unique barcode identification
Solution Approach 2:
Each microdeletion detection component within the multiplex assay has localized quality characteristics through unique barcode sequences. This local differentiation allows the system to maintain high reliability for each specific microdeletion type while achieving multiplexing capability, as each barcode provides distinct identification that prevents false positives even when multiple assays are performed simultaneously
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
Enhances the accuracy and efficiency of detecting microdeletions by providing a positive control for PCR assays, determining efficiency and error rates, and enabling non-invasive prenatal testing for subchromosomal abnormalities.
Implementation Method 1
performing targeted amplification on the spiked sample or fractions thereof from (b) to amplify one or more target regions comprising microdeletions of interests to obtain amplicons
Data Source
AI summary
Provided herein are compositions to be used as a positive control for detection of one or more microdeletions of interest in a sample. The positive control can be used to determine an error and an efficiency rate for assays used to identify microdeletions such as 22q1 1.2 deletion (DiGeorge syndrome), chromosome 5p15.2 (Cri-du-chat), 1p36 deletion, 15q1 1.2˜q13 deletion (Prader-Willi syndrome), and/or 15q1 1˜q13 (Angelman syndrome).