Nucleic Acid Sequence Element Detection via Segmented Amplification
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Solution Overview
Problem
Current methods for processing biological samples often fail to accurately detect sequence elements due to sub-optimal placement of identifying sequences or interference during sample processing, leading to confounded analysis of exogenous nucleic acid molecules.
Innovation Solution
A method involving the amplification of target nucleic acid sequences using specific primers, fragmentation, and detection of nucleic acid fragments, with optional barcoding and sequencing, to establish the presence of sequence elements within biological samples, particularly in partitioned environments like droplets or wells.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If identifying sequence elements are placed in exogenous nucleic acid molecules for detection, then the presence of exogenous nucleic acid can be identified, but the analysis is confounded if the sequence elements are located sub-optimally or if processing precludes their detection
Solution Approach 1:
The method divides the detection process into two independent stages: (1) amplification of the entire exogenous nucleic acid molecule using primers that anneal to the sequence element, and (2) detection of a fragment generated from the amplification product. This segmentation ensures that the sequence element is captured during amplification while the actual detection can be performed on a fragment that is optimized for detection methods, resolving the contradiction between identification and reliable analysis
Solution Approach 2:
The method extracts the sequence element information indirectly by amplifying it with specific primers and then detecting a fragment that contains or is derived from this amplified region. This extraction approach allows the detection system to focus on a manageable fragment while the amplification step ensures comprehensive capture of the target sequence, thereby improving both detection accuracy and analysis reliability
2Ease of operation
If standard amplification and detection methods are used, then the process is simple, but they fail to detect sequence elements when sub-optimally placed or when processing interferes
Solution Approach 1:
The method performs preliminary amplification of the exogenous nucleic acid molecule using primers specifically designed to anneal to the sequence element before detection. This preliminary action ensures that even if the sequence element is sub-optimally placed or obscured during sample processing, it will be faithfully replicated and made accessible for subsequent detection, thereby maintaining both process simplicity and detection accuracy
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 method enables precise identification and analysis of sequence elements, overcoming previous limitations by ensuring accurate detection and analysis of exogenous nucleic acid molecules, even in complex sample environments.
Implementation Method 1
a first amplification of a target nucleic acid molecule sequence with aid of a first primer annealed to the sequence element in the target nucleic acid molecule sequence
Implementation Method 2
amplification of a target nucleic acid sequence using primers designed to anneal to a sequence element
Implementation Method 3
fragmenting the amplified nucleic acid molecule, thereby generating a nucleic acid fragment
Implementation Method 4
the detecting establishes the presence of the sequence element in the target nucleic acid molecule sequence
Data Source
AI summary
Provided herein are methods and systems for establishing the presence of a sequence element in nucleic acid molecules. The sequence element may comprise a fused gene, a reporter gene, or another useful sequence for cell and tissue engineering, such as those used for labeling cells, identifying successfully transfected or transduced cells, etc. A method provided herein may additionally allow for barcoding of nucleic acid molecules and analysis of libraries of barcoded nucleic acid molecules.