Self-Complementary MDA Primers for Bias-Reduced Pooled Sequencing
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Solution Overview
Problem
Current multiple displacement amplification (MDA) methods face challenges with amplification bias and require individual processing of samples due to the inability to identify samples until late in the procedure, limiting throughput and increasing costs, especially in large-scale nucleic acid sequencing.
Innovation Solution
The use of primers with self-complementary sequences at the 5′ terminus and random or semi-random sequences at the 3′ terminus for MDA reactions, allowing for sample pooling and identification through unique cell index sequences, reducing amplification bias and enabling high-throughput sequencing.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Loss of information
If regular MDA primers are modified by adding defined sequences at their 5′ termini to serve as cell index sequences, then sample identification capability is improved, but amplification artefacts and bias increase
Solution Approach 1:
The invention extracts the cell index sequence from the primer's functional region (where it would bind to target DNA) and places it at the 5′ terminus. This separation allows the index sequence to serve its identification purpose without interfering with the random sequence's ability to bind randomly to genomic DNA, thereby maintaining amplification uniformity while enabling sample identification.
Solution Approach 2:
The invention introduces a self-complementary sequence as an intermediary element at the 5′ terminus of the primer. This self-complementary sequence forms a hairpin structure that prevents the cell index sequence from interacting with target DNA during amplification, acting as a mediator that allows the index sequence to be present without causing amplification bias or artifacts.
2Reliability
If random sequences of 6-10 bases are used as primers in MDA reactions, then amplification potency and reduction of bias are improved, but sample identification capability deteriorates requiring individual processing
Solution Approach 1:
The invention merges two previously separate functions into a single primer molecule: (1) the random sequence at the 3′ terminus that binds to genomic DNA and initiates amplification, and (2) the cell index sequence at the 5′ terminus that provides sample identification. This combination allows multiple samples to be processed together in pooled MDA reactions while maintaining both amplification quality and sample traceability.
3Reliability
If individual sample processing is performed in MDA reactions, then amplification quality is maintained, but preparation costs and time increase substantially
Solution Approach 1:
The invention performs preliminary action by incorporating unique cell index sequences into the primers before the MDA reaction. This allows samples to be pre-pooled and processed together in a single MDA reaction rather than requiring individual processing followed by identification at a later stage, thereby reducing both time and cost while maintaining amplification quality through the self-complementary sequence design.
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 minimizes amplification bias, facilitates sample pooling, and reduces preparation costs, enhancing sequencing error corrections and throughput in nucleic acid sequencing workflows.
Implementation Method 1
a primer set, wherein each primer of the primer set comprises a self-complementary sequence at its 5′ terminus
Implementation Method 2
a DNA polymerase having a strand displacement activity
Implementation Method 3
Multiple Displacement Amplification (MDA) using a DNA polymerase with a strand displacement activity under isothermal conditions
Implementation Method 4
a random sequence or a semi-random sequence at its 3′ terminus
Data Source
AI summary
The present disclosure provides primers, primer sets, kits and methods for multiple displacement amplification, especially in combination with nucleic acid sequencing. The primers comprise self-complementary sequences at their 5′ termini and random or semi-random sequences at their 3′ termini. Use of such primers facilitates handling of multiple samples, increases sequence coverage uniformity, and improves sequencing error corrections.