Chimeric Amplicon Formation via Primer Stopper Template Switching
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Solution Overview
Problem
Current PCR and ligation methods for sequencing library preparation face limitations such as low efficiency in appending adapter sequences, requiring multiple thermo-cycling reactions, and being restricted by thermostable polymerases, leading to loss of molecules and inefficiencies in forming chimeric amplicons.
Innovation Solution
The use of specifically designed Primer and Stopper oligonucleotides that induce template switching during polymerase extension, allowing for the formation of chimeric amplicons with sequences inherited from both Target and Stopper nucleic acid molecules, using a single isothermal polymerase extension step, and compatible with both thermostable and non-thermostable polymerases.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If ligation method is used to append adapter sequences, then adapter sequences can be appended to nucleic acid molecules, but the efficiency is low (10-30%) leading to loss of majority of molecules
Solution Approach 1:
The patent replaces the ligation enzymatic mechanism with a polymerase extension mechanism. Instead of using ligase to join adapter sequences to nucleic acid molecules (which has low efficiency), the invention uses a polymerase to extend primers along the template, incorporating adapter sequences efficiently. This substitution of the biochemical mechanism achieves near 100% efficiency in adapter appending while eliminating the 70-90% molecule loss associated with ligation.
2Productivity
If PCR method is used to append adapter sequences to both ends of amplicons, then adapter sequences can be appended to both ends, but at least two cycles are required and thermostable polymerase is needed
Solution Approach 1:
The patent incorporates adapter sequences directly into the primer designs before the extension reaction begins. The forward primer contains the reverse complement of the forward adapter sequence, and the reverse primer contains the reverse complement of the reverse adapter sequence. This preliminary incorporation of adapter sequences into primers allows both ends of the amplicon to receive adapters in a single extension cycle, eliminating the need for multiple PCR cycles and the requirement for thermostable polymerases.
3Productivity
If thermostable polymerase and thermo-cycling reactions are used, then adapter sequences can be appended, but the method is restricted by these requirements
Solution Approach 1:
The patent creates a universal adapter appending system that works with any polymerase capable of extending DNA primers, not just thermostable polymerases. By using a single extension reaction at the optimal temperature for the chosen polymerase (which could be room temperature, 37°C, or any other temperature depending on the polymerase used), the method eliminates the restriction to thermostable polymerases and thermo-cycling. This makes the system adaptable to various polymerases including non-thermostable ones, expanding versatility while maintaining the ability to append adapters to both ends of amplicons.
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 achieves high-yield template switching, up to 100% efficiency, overcoming the limitations of traditional methods by enabling adapter sequence appending to both ends of amplicons in a single step, independent of thermo-cycling, and improving the formation of chimeric sequences.
Implementation Method 1
forming a chimeric amplicon of a Target nucleic acid by polymerase extension
Implementation Method 2
the Third Sequence is complementary to a Match Region sequence positioned to the 3′ of the Binding Region on the Target nucleic acid
Data Source
AI summary
Provided herein are compositions and methods for formation of amplicons having a chimeric sequence, partially derived from a target nucleic acid and partially derived from a rationally designed oligonucleotide. The provided compositions provide for high-yield, induced template switching between the target nucleic acid and the rationally designed oligonucleotide as the template during polymerase extension, achieving inheritance of information from both within only one cycle.