5'-5' Linked Oligonucleotides for Long DNA Sequencing
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Solution Overview
Problem
Current methods for simultaneous detection, sequencing, and cloning of multiple different polynucleotides, such as the ends of long DNA molecules or different mRNA molecules, are inefficient, costly, and suffer from high error rates, especially when dealing with DNA sequences longer than 5 kb.
Innovation Solution
The use of 5'-5' linked oligonucleotides, referred to as chain-seq or crab-seq oligos, which comprise two or more arms linked at the 5' end, allowing for the covalent linkage and amplification of nucleic acid molecules, enabling the creation of a single amplicon that incorporates the sequence of both target molecules, facilitating further processing like sequencing.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If conventional methods are used for simultaneous detection and sequencing of multiple different polynucleotides, then the process is simple, but the efficiency is low, cost is high, and error rate is high
Solution Approach 1:
The invention segments the detection process by segregating nucleic acid molecules into individual discrete volumes (droplets), allowing independent processing of each molecule. This segmentation enables simultaneous detection of multiple polynucleotides while maintaining high accuracy through isolated amplification events, directly resolving the contradiction between reliability and productivity.
Solution Approach 2:
The invention merges multiple nucleic acid molecules into a single complex through covalent linkage of primers to the target molecules. By combining multiple segregated molecules into one linked complex that can be processed together, the method achieves both high accuracy (through individual segregation) and high efficiency (through combined processing), resolving the contradiction between reliability and productivity.
2Reliability
If conventional sequencing methods are used for long DNA molecules (>5 kb), then the process is straightforward, but the error rate increases and efficiency decreases
Solution Approach 1:
The invention performs preliminary segregation of nucleic acid molecules into individual discrete volumes before amplification and sequencing. This pre-processing step ensures that each long DNA molecule is isolated and properly prepared in advance, preventing errors that would accumulate during processing and reducing overall processing time by enabling parallel化处理, thus resolving the contradiction between accuracy and processing time.
3Productivity
If multiple different polynucleotides are processed simultaneously using conventional methods, then the complexity is low, but the cost increases and efficiency decreases
Solution Approach 1:
The invention employs universal primers and reagents that can bind to and process multiple different types of polynucleotides (DNA, RNA, cDNA) through the same covalent linkage mechanism. This multi-functionality allows simultaneous processing of diverse nucleic acid molecules with a single standardized protocol, increasing efficiency while keeping method complexity manageable, thus resolving the contradiction between productivity and device complexity.
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 efficiency and accuracy of sequencing long DNA reads, reduces costs, and improves the handling of large DNA inputs by generating a single amplicon that can be used for downstream applications like de novo genome assembly and mutation detection.
Implementation Method 1
annealing, within each individual discrete volume, said two or more nucleic acid molecules using a first primer and a second primer, wherein the first and second primers are linked by a 5'-5'-covalent linkage, and wherein the first primer hybridizes to a first sequence of a first nucleic acid molecule
Implementation Method 2
amplifying the first and the second nucleic acid molecules in the complex with a reverse transcriptase under conditions to create a first cDNA complementary to the first nucleic acid molecule and a second cDNA complementary to the second nucleic acid molecule
Implementation Method 3
amplifying the first and the second nucleic acid molecules in the complex under conditions to create a first DNA complementary to the first nucleic acid molecule and a second DNA complementary to the second nucleic acid molecule
Data Source
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AI summary
The present disclosure generally relates to methods and compositions of linking, amplifying, and sequencing nucleic acid molecules. Also disclosed is the use of 5'-5'linked oligonucleotides for linking nucleic acid molecules for sequencing of the ends of long nucleic acid template molecules, or for sequencing polymorphism or different target genes or different RNAs simultaneously.