Nucleic Acid Amplification Bias Reduction via Ligation
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Solution Overview
Problem
Existing nucleic acid amplification methods, such as PCR and isothermal techniques, often suffer from amplification bias when amplifying short nucleic acid sequences like microRNAs, which can lead to variations based on sequence and abundance, making it challenging to accurately determine the relative amounts of multiple target nucleic acids in a sample.
Innovation Solution
A method involving the ligation of two or more different target nucleic acids to form a ligated product, which is then amplified, allowing for the simultaneous amplification of multiple targets while reducing amplification bias by using adaptor and primer sequences with specific GC content and lengths, and subsequently determining the relative amounts through quantitative real-time PCR.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If conventional amplification methods (PCR or isothermal techniques) are used to amplify multiple target nucleic acids, then amplification can be achieved, but amplification bias occurs due to variations in sequence and abundance
Solution Approach 1:
The patent merges multiple different target nucleic acids into a single ligated product by ligating them together with adaptor sequences. This combining approach allows simultaneous amplification of multiple targets in one reaction, improving productivity while the ligated structure ensures proportional representation, thereby maintaining measurement precision.
Solution Approach 2:
The patent introduces adaptor sequences as intermediary elements that ligate multiple target nucleic acids together. These adaptors serve as mediators that enable simultaneous amplification while preserving the relative abundance information of each target, thus resolving the contradiction between amplification capability and measurement accuracy.
2Measurement precision
If multiple target nucleic acids are amplified individually, then each target can be amplified, but the process is time-consuming and inefficient
Solution Approach 1:
The patent combines multiple individual amplification processes into a single simultaneous amplification reaction by ligating multiple targets into one product. This merging eliminates the need for separate amplification steps for each target, dramatically reducing time loss while maintaining the ability to accurately measure each individual target's relative amount through quantitative real-time PCR.
3Productivity
If amplification is performed on short nucleic acid sequences like microRNAs, then amplification can occur, but amplification bias varies according to sequence composition (AT content or GC content)
Solution Approach 1:
The patent uses adaptor sequences as intermediaries that ligate to the ends of short nucleic acid sequences. These adaptors provide standardized ends that facilitate efficient ligation and amplification while the ligated structure ensures that amplification efficiency is normalized across different sequences, reducing bias related to AT or GC content variations.
Solution Approach 2:
The patent changes the structural parameters of short nucleic acid sequences by ligating them into larger constructs with standardized adaptor sequences. This parameter change (from short individual sequences to ligated products with adaptors) improves amplification productivity while the standardized structure reduces amplification bias across different sequence compositions.
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 the accurate and proportional amplification of multiple target nucleic acids, reducing amplification bias and allowing for precise determination of their relative amounts, thereby improving the accuracy of nucleic acid profiling in samples.
Implementation Method 1
The amplification of a nucleic acid includes methods that require multiple cycles during the amplification process, and methods that are performed at a single temperature. Cycling techniques are exemplified by methods requiring thermo-cycling, such as polymerase chain reaction (PCR).
Implementation Method 2
PCR generally involves thermally denaturing a double-stranded DNA to provide single stranded DNAs
Implementation Method 3
annealing a primer to the single stranded DNA; and synthesizing a complementary strand from the primer
Implementation Method 4
the isothermal techniques rely on a strand displacing polymerase in order to separate the two strands of a double strand and re-copy the template
Data Source
AI summary
A method of amplifying a target nucleic acid with reduced amplification bias and a method of determining a relative amount of a target nucleic acid in a sample.