Single DNA Molecule Isolation for Mixed Sample Resolution
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Solution Overview
Problem
Current methods for analyzing DNA mixtures are limited by spurious sequence content introduced during DNA amplification and sequencing, which prevents the detection of minor contributors below 5% in forensic samples, necessitating the development of techniques that can eliminate noise sources and enable detection down to the 1/1000 level.
Innovation Solution
The method involves isolating single DNA molecules prior to amplification and applying unique barcoded primers to tag spurious allelic sequences, allowing for error-free sequencing by selecting the majority sequence for each molecule, thereby eliminating noise sources and enabling the detection of minor contributors through droplet microfluidic devices and bioinformatics analysis.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If conventional DNA amplification and sequencing methods are used, then standard forensic analysis can be performed, but spurious sequence content is introduced that limits detection of minor contributors to above 5%
Solution Approach 1:
The patent segments the DNA analysis process by isolating individual DNA molecules into separate droplets before amplification. Each droplet contains at most one DNA molecule, which is then amplified with unique barcoded primers. This segmentation prevents spurious sequence content from different molecules from being mixed together, enabling detection of minor contributors at the 1/1000 level by analyzing each molecule's sequence independently and selecting the majority sequence for each barcode.
2Reliability
If differential extraction and computational analysis of allele peak heights are used, then mixed contributor samples can be analyzed, but the method requires specific assumptions and cannot resolve contributors below 5% with certainty
Solution Approach 1:
The patent replaces the mechanical/electrical system of capillary electrophoresis and peak height analysis with a molecular tagging system using DNA barcodes. Instead of measuring allele peak heights and making computational assumptions, each DNA molecule is tagged with a unique barcode during amplification, allowing direct sequencing and identification. This substitution eliminates the need for differential extraction and complex computational analysis, providing certain resolution of minor contributors at the 1/1000 level through direct molecular identification.
3Productivity
If next generation DNA sequencing is used, then more reads are obtained, but spurious content from amplification and base calling errors still limits resolution to above 5%
Solution Approach 1:
The patent introduces unique barcoded primers as intermediaries between the DNA template and the sequencing process. Each primer contains a unique barcode that is incorporated during amplification, serving as a molecular identifier. This intermediary system allows the sequencing machine to group reads by their originating DNA molecule, enabling the selection of majority sequences for each barcode and eliminating spurious content from amplification and base calling errors, thereby maintaining high precision even with high throughput sequencing.
Data Source
AI summary
Methods for analyzing DNA-containing samples are provided. The methods can comprise isolating a single genomic equivalent of DNA from the DNA-containing sample to provide a single isolated DNA molecule. The single isolated DNA molecule can be subjected to amplification conditions in the presence of one or more sets of unique molecularly tagged primers to provide one or more amplicons. Any spurious allelic sequences generated during the amplification process are tagged with an identical molecular tag. The methods can also include a step of determining the sequence of the one or more amplicons, in which the majority sequence for each code is selected as the sequence of the single original encapsulated target. The DNA-containing sample can be a forensic sample (e.g., mixed contributor sample), a fetal genetic screening sample, or a biological cell.