Engineered Templates for Single Primer Antibody Amplification
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Solution Overview
Problem
Traditional nucleic acid amplification methods, such as PCR, require two gene-specific primers and are prone to non-specific amplification, especially when amplifying antibody genes, leading to skewed diversity and loss of rare genes due to optimal annealing temperature issues.
Innovation Solution
The development of methods using engineered templates created through nick ligation and polymerase extension, which allow for specific amplification of antibody genes without gene-specific primers, enabling the addition of predetermined sequences to polynucleotides using restriction endonucleases and ligase, facilitating single primer amplification.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If traditional PCR with two gene-specific primers is used, then amplification can be performed, but non-specific amplification occurs and specificity is reduced
Solution Approach 1:
The patent applies preliminary action by pre-modifying the template DNA with adapter sequences before amplification. The template preparation step includes adding adapter sequences that enable universal primer binding, so that when amplification occurs, the primers bind to these predetermined adapter regions rather than requiring gene-specific sequences, thereby preventing non-specific amplification before it can occur
Solution Approach 2:
The patent uses adapter sequences as intermediaries between the template DNA and the primers. These adapter sequences serve as a mediator that allows universal primers to bind to the template without requiring gene-specific sequences, thus enabling specific amplification through a intermediary structure rather than direct primer-template matching
2Productivity
If forward primers anneal to framework 1 region to accommodate somatic mutations, then amplification efficiency improves, but reverse primers annealing to constant region causes non-specific amplification
Solution Approach 1:
The patent applies preliminary action by pre-modifying the template DNA with adapter sequences before amplification. The template preparation step includes adding adapter sequences that enable universal primer binding, so that when amplification occurs, the primers bind to these predetermined adapter regions rather than requiring gene-specific sequences, thereby preventing non-specific amplification before it can occur
Solution Approach 2:
The patent applies universality by using universal primers that can bind to all template sequences through the adapter regions. The adapter sequences serve as a universal binding site for all primers, allowing the same primer to amplify different gene sequences without requiring gene-specific optimization, thus achieving both efficiency and specificity
3Productivity
If two gene-specific primers are used for PCR, then amplification can proceed, but optimal annealing temperature cannot be achieved for both primers simultaneously
Solution Approach 1:
The patent applies universality by using universal primers that can bind to all template sequences through the adapter regions. The adapter sequences serve as a universal binding site for all primers, allowing the same primer to amplify different gene sequences without requiring gene-specific optimization, thus achieving both efficiency and specificity
Solution Approach 2:
The patent applies parameter changes by shifting the binding location from gene-specific regions to adapter regions. This changes the annealing temperature parameter from being gene-dependent to being primer-dependent, allowing standardized thermal cycling conditions to be used across different gene amplifications
4Productivity
If transcription-based amplification is used, then amplification can occur, but polymerase promoter sequence incorporation by primer leads to non-specific amplification
Solution Approach 1:
The patent uses adapter sequences as intermediaries between the template DNA and the primers. These adapter sequences serve as a mediator that allows universal primers to bind to the template without requiring gene-specific sequences, thus enabling specific amplification through a intermediary structure rather than direct primer-template matching
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
These methods enhance the specificity and diversity of amplified gene repertoires by allowing for the precise addition of sequences, reducing non-specific amplification and improving the detection of rare genes, thereby improving the accuracy and efficiency of nucleic acid amplification.
Implementation Method 1
annealing an oligonucleotide to a polynucleotide
Implementation Method 2
the oligonucleotide and the polynucleotide are ligated in the presence of a ligase
Implementation Method 3
the polynucleotide is cleaved at a restriction site
Data Source
AI summary
This disclosure relates to methods for creating engineered templates that are useful for amplification of one or more antibody genes without the use of gene-specific primers. More specifically, templates engineered using these methods in a polymerase chain reaction setting which allows for the specific amplification of one or more antibody genes.