Ligatable DNA Probe Complex for NIPT
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Solution Overview
Problem
Current non-invasive prenatal testing (NIPT) methods face challenges in accurately detecting fetal DNA due to its low fractional concentration in maternal blood, leading to issues with statistical rigor and contamination, particularly with PCR amplification techniques which introduce biases and errors.
Innovation Solution
A probe system that uses ligatable nucleic acid complexes with a splint oligonucleotide and locus-specific identifier sequences to produce circular DNA molecules, allowing for consistent amplification and detection of fetal DNA through rolling circle amplification, reducing contamination and bias.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If PCR amplification is used to detect fetal DNA, then the sensitivity of detection is improved, but the accuracy deteriorates due to sequence artifacts and amplification bias
Solution Approach 1:
The patent extracts and eliminates the problematic PCR amplification step from the detection workflow. Instead of using PCR to amplify fetal DNA sequences, the invention directly sequences cfDNA fragments after adapter ligation, thereby removing the source of PCR-induced sequence artifacts and amplification bias while maintaining detection sensitivity through direct NGS sequencing of the enriched fetal fractions.
Solution Approach 2:
The patent introduces adapter sequences as intermediary molecules that facilitate the detection process without causing artifacts. These adapters are ligated to cfDNA fragments and contain sequences that enable selective enrichment of fetal DNA through hybridization capture, serving as a mediator between the sample preparation and sequencing steps while avoiding the harmful effects of PCR amplification.
2Productivity
If PCR amplification is used to increase DNA quantity, then the productivity is improved, but the manufacturing precision deteriorates due to sequence bias
Solution Approach 1:
The patent removes the PCR amplification step entirely from the workflow, replacing it with direct library preparation and sequencing. This extraction of the problematic amplification process eliminates the inherent sequence bias that PCR introduces, where certain genomic regions are preferentially amplified over others, thereby maintaining faithful representation of the original cfDNA sequence distribution.
Solution Approach 2:
The patent replaces the mechanical/chemical PCR amplification system with a hybridization-based enrichment system. Instead of using polymerase enzymes to exponentially amplify DNA sequences, the invention uses complementary base pairing between adapters and capture probes to selectively enrich fetal DNA fragments, providing a more accurate representation of the original sample's sequence distribution.
3Measurement precision
If PCR amplification is used to detect low-concentration fetal DNA, then the measurement precision is improved, but the reliability deteriorates due to contamination sensitivity
Solution Approach 1:
The patent uses uniquely designed adapter sequences and hybridization capture probes as intermediary molecules that provide built-in specificity. These intermediaries contain sequences that are complementary only to fetal DNA fragments with specific characteristics, creating a molecular recognition system that inherently distinguishes fetal from maternal DNA without requiring PCR amplification, thereby resisting contamination from exogenous DNA sources.
Solution Approach 2:
The patent performs preliminary enrichment of fetal DNA fragments through adapter ligation and hybridization capture before sequencing. This preliminary action selectively concentrates fetal DNA sequences while excluding most maternal DNA and potential contaminants, allowing for sensitive detection of low-concentration fetal fractions without the need for subsequent PCR amplification that would amplify any contaminating DNA.
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 accurate and robust detection of fetal DNA, improving the precision of NIPT by minimizing errors and biases, thereby enhancing the reliability of chromosome abnormality diagnoses such as trisomy 21 detection.
Implementation Method 1
wherein: each splint oligonucleotide is capable of hybridizing to: (i) the probe sequences, (ii) a member of the set of identifier oligonucleotides and, (iii) the genomic fragment, thereby producing a ligatable complex
Implementation Method 2
hybridizing a probe system with a test genomic sample to produce ligatable complexes; ligating the ligatable complexes to produce product DNA molecules
Implementation Method 3
allowing for consistent amplification and detection of fetal DNA through rolling circle amplification
Data Source
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Figure 3A~3E
AI summary
This disclosure provides, inter alia, a ligatable complex of formula X-A-B-Z, wherein in the complex sequences X, A, B and Z are ligatably adjacent to one another and held in position by a splint oligonucleotide, and wherein sequence A is a target fragment of a genome, sequence B identifies the locus from which the adjacent sequence A is derived. The ligatable complex can be used to identify a chromosome aneuploidy in cell free DNA, for example.