Unidirectional Primer Extension Amplification via Covalently-Closed Circle
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Solution Overview
Problem
Existing methods for amplifying unidirectional primer extension products in next-generation sequencing require additional steps to create a reverse PCR primer binding target, which are often inefficient.
Innovation Solution
The method involves creating a covalently-closed circle structure on the initial unidirectional primer extension product, allowing both forward and reverse PCR primer binding targets to be present, facilitating amplification through inverse PCR or rolling circle amplification.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If traditional PCR is used for amplification, then amplification can be performed, but additional steps are required to create a reverse PCR primer binding target which reduces efficiency
Solution Approach 1:
The bridge oligonucleotide is incorporated into the probe structure beforehand, so that when the probe hybridizes to the target and undergoes primer extension, the bridge oligonucleotide is automatically positioned to form the reverse PCR primer binding target. This preliminary arrangement eliminates the need for separate target creation steps.
Solution Approach 2:
The probe structure merges three functional elements: the target-binding unique region, the constant region for PCR primer binding, and the bridge oligonucleotide that creates the reverse PCR primer binding target. This consolidation of functions into a single probe molecule streamlines the amplification process.
2Productivity
If blunt-ended ligation, single stranded DNA ligation or homopolymer tailing are used to create reverse PCR primer binding target, then amplification can proceed, but these approaches are often inefficient
Solution Approach 1:
The invention extracts the function of creating the reverse PCR primer binding target from separate post-extension processing steps and integrates it directly into the probe structure itself. The bridge oligonucleotide component is designed to automatically form the binding target during the extension reaction, eliminating inefficient separate ligation or tailing steps.
3Device complexity
If rolling-circle amplification is used, then only a single primer binding target is required, but the template molecule must exist as a covalently closed circle structure which requires additional preparation
Solution Approach 1:
The probe structure is preliminarily designed with the bridge oligonucleotide sequence that is complementary to the constant region. When the probe hybridizes to the target and extension occurs, the bridge oligonucleotide automatically forms a hanging end that can anneal back to the constant region, creating the circular structure needed for rolling-circle amplification without requiring separate circularization steps.
4Adaptability or versatility
If de novo creation of reverse PCR primer binding target is required, then traditional PCR can be used, but the sequence is unknown which complicates the process
Solution Approach 1:
The constant region of the probe serves multiple functions: it provides a binding site for the forward PCR primer, contains the sequence for the reverse PCR primer binding target via the bridge oligonucleotide, and enables both traditional PCR and rolling-circle amplification. This multi-functionality eliminates the need for de novo sequence determination.
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 enables efficient amplification of unidirectional primer extension products with minimal sample requirements, as low as 10ng of template, and allows for the identification of sequence variants at sub-Mendelian allele frequencies.
Implementation Method 1
annealing one or more probes to the one or more target nucleic acid molecules in the sample to form one or more probe-target nucleic acid molecule complexes, wherein the unique region of each probe includes a nucleotide sequence which hybridizes to a complementary nucleic acid sequence within the one or more target nucleic acid molecules
Implementation Method 2
extending the one or more probe-target nucleic acid molecule complexes to form one or more extended probe-target nucleic acid molecule complexes
Implementation Method 3
generating one or more ligation products from the one or more extended probe-target nucleic acid molecule complexes
Data Source
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AI summary
The present disclosure is directed to compositions, kits, and methods of and methods which facilitate the amplification of a unidirectional primer extension product. In particular, the compositions, kits, and methods described herein facilitate the amplification of a unidirectional primer extension product without the need to incorporate a second polymerase chain reaction primer binding target on a distal end of an initial single-stranded nucleic acid molecule primer extension product.