Emulsion Microfluidics Sequencing via Primer Partitioning
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Solution Overview
Problem
Current DNA sequencing methods face challenges in accurately determining nucleotide sequences due to limitations in primer specificity and efficiency in hybridization reactions, particularly in distinguishing between fully complementary and non-complementary primers.
Innovation Solution
The method involves partitioning a target nucleic acid mixture with a series of primers, each having multiple designated nucleotides, and combining them in reaction partitions to hybridize under specific conditions, where fully complementary primers bind and non-complementary ones do not, allowing for the determination of the nucleotide sequence based on primer binding.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If conventional DNA sequencing methods are used with standard primers, then the sequencing process can be performed, but the accuracy of nucleotide sequence determination is reduced due to inability to distinguish fully complementary primers from non-complementary primers
Solution Approach 1:
The patent segments the hybridization process into distinct phases: first hybridization with family-specific primers, then second hybridization with extended primers containing additional designated nucleotides. This segmentation allows progressive refinement of sequence determination, improving accuracy by eliminating ambiguous bindings at each stage
Solution Approach 2:
The patent applies local quality by designing primers with specific regions of high specificity (fully complementary designated nucleotides) combined with regions of controlled degeneracy (degenerate bases or universal nucleotides). This local differentiation enables the primer to bind selectively to fully complementary sequences while rejecting mismatched sequences, thereby improving measurement precision
2Loss of information
If multiple primers are used in hybridization reactions, then more sequence information can be obtained, but the efficiency of hybridization reactions decreases due to difficulty in distinguishing specific bindings
Solution Approach 1:
The patent divides the primer set into multiple families, each targeting specific sequence regions. By segmenting the hybridization process into multiple targeted reactions rather than one large multiplex reaction, the system maintains high efficiency while obtaining comprehensive sequence information across the entire target region
Solution Approach 2:
The patent employs a feedback mechanism where hybridization results from first-round primers inform the selection and design of second-round extended primers. This feedback loop allows the system to efficiently focus subsequent hybridization reactions on regions requiring further resolution, optimizing both information gain and reaction efficiency
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 accuracy and efficiency of nucleotide sequence determination by ensuring only fully complementary primers hybridize, thereby resolving the sequence through the presence or absence of fluorescent signals, and deconvoluting the sequence information from hybridization data.
Implementation Method 1
hybridizing the target nucleic acid to the primers in the reaction partitions under conditions in which fully complementary primers hybridize to the target sequence and primers that are not fully complementary do not hybridize to the target nucleic acid
Implementation Method 2
the target nucleic acid comprises a fluorescent moiety and is annealed to a quenching oligonucleotide that comprises a quencher, wherein annealing of the quencher to the target nucleic acid quenches fluorescence of the fluorescence moiety
Implementation Method 3
contacting the target nucleic acid annealed to the quencher oligonucleotide with a primer-dependent polymerase, wherein extension of the primers, if hybridized, results in displacement of the quencher oligonucleotide, thereby generating a fluorescent signal
Data Source
AI summary
Methods, libraries, and kits for nucleotide sequencing are provided. For example, methods can comprise providing a plurality of partitions, the partitions comprising a set of two or more different primers and a target nucleic acid, wherein different partitions contain different sets of primers with a majority of primers in the sets being common with a different primer set but wherein any two different partitions having a common primer contain no more than one common primer; and hybridizing the target nucleic acid to the primers in the reaction partitions under conditions in which fully complementary primers hybridize to the target sequence and primers that are not fully complementary do not hybridize to the target nucleic acid.