Transposome-Based Polynucleotide Tagmentation and Extension for High-Yield Sequencing
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Solution Overview
Problem
Existing methods for preparing nucleic acid samples for sequencing, such as transposase-mediated fragmentation and tagging, often suffer from low yields and non-specific amplification due to primer dimer products.
Innovation Solution
A method involving contacting a target polynucleotide with transposomes to yield tagged fragments, followed by an extension reaction using specific primers, and subsequent amplification with a primer pair that includes a transposon sequence or its complement, to produce amplification products with improved yield and specificity.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Device complexity
If transposase-mediated fragmentation and tagging is used to simplify sample preparation, then workflow complexity is reduced, but amplification yield decreases and non-specific amplification increases
Solution Approach 1:
The patent divides the amplification process into distinct stages: tagmentation to generate tagged fragments, extension reaction to create extension products, and final amplification. This segmentation allows optimization of each stage independently, improving overall yield while maintaining workflow simplicity.
Solution Approach 2:
The patent introduces extension primers and extension products as intermediary molecules between the tagged fragments and final amplification products. These intermediaries serve as bridges that enable specific amplification while reducing non-specific products, thus improving yield without complicating the workflow.
2Device complexity
If transposase-mediated fragmentation and tagging is used, then sample preparation is simplified, but specificity of amplification decreases due to primer dimer products
Solution Approach 1:
The patent designs extension primers with specific local characteristics: a 3' end segment that is complementary to the tagged fragment and a 5' end segment that lacks complementarity. This local differentiation ensures specific binding to target sequences while preventing primer dimer formation, thereby improving amplification specificity.
Solution Approach 2:
The patent modifies primer parameters by designing extension primers with asymmetric complementarity - full complementarity at the 3' end for specific binding and no complementarity at the 5' end to prevent self-annealing. This parameter change eliminates primer dimer products while maintaining simplified sample preparation.
3Loss of time
If existing amplification methods are used, then sample preparation is rapid, but yield is low due to non-specific amplification
Solution Approach 1:
The patent performs tagmentation and extension reactions as preliminary actions before final amplification. The extension reaction prepares specific extension products that serve as templates for high-yield amplification, ensuring both rapid preparation and high yield by eliminating non-specific products in advance.
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 enhances the yield and specificity of amplification products, reducing non-specific amplification and improving the efficiency of sample preparation for sequencing applications.
Implementation Method 1
contacting a target polynucleotide present in a polynucleotide sample with transposomes to yield a plurality of tagged fragments, individual transposomes comprising a transposase complexed with a transposon sequence
Implementation Method 2
subjecting the plurality of tagged fragments to an extension reaction using extension primers to yield extension products, individual extension primers having a segment at a 3' end exhibiting sequence complementarity to a tagged fragment
Implementation Method 3
amplifying the extension products using a primer pair to yield amplification products, the primer pair including a first primer comprising the transposon sequence or a portion thereof and a second primer comprising the sequence of the segment at the 5' end of the extension primer
Data Source
AI summary
Provided herein are methods, compositions, and kits for forming amplification products. In various embodiments provided herein, transposomes comprising transposases are used in forming tagged polynucleotides for downstream amplification and polynucleotide processing steps.