Self-Complementary MDA Primers for Low-Bias Sample Pooling
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Solution Overview
Problem
Existing 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
Utilizing primers with self-complementary sequences at the 5′ terminus and random or semi-random sequences at the 3′ terminus for MDA reactions, allowing for separate reactions to be pooled and identified by unique cell index sequences, reducing bias and enabling high-throughput sample preparation.
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 primer is divided into two functional segments: a self-complementary sequence segment at the 5′ terminus that forms a hairpin structure, and a random sequence segment at the 3′ terminus that binds to the template. This segmentation allows the cell index function to be isolated in the hairpin structure, preventing it from interfering with the amplification process while still providing sample identification capability.
Solution Approach 2:
The defined sequence at the 5′ terminus is designed to be self-complementary, changing its structural parameter from a linear sequence to a hairpin structure. This parameter change prevents the defined sequence from annealing to the template in a way that would cause bias, while maintaining its function as a cell index.
2Reliability
If separate MDA reactions are performed for each sample, then amplification bias is reduced, but sample throughput is limited and preparation costs increase
Solution Approach 1:
The self-complementary sequence serves multiple functions: it enables sample identification through the hairpin structure, allows for pooling of multiple samples in a single reaction, and maintains reduced amplification bias. This multi-functionality resolves the contradiction between handling samples separately and achieving high throughput.
Solution Approach 2:
The self-complementary sequence acts as an intermediary that enables sample identification without requiring separate processing. It mediates between the need for individual sample handling and the desire for high-throughput pooled processing by providing a molecular tag that works in both contexts.
3Power
If random sequences of 6-10 bases are used as primers in MDA, then amplification potency is maintained, but individual sample identification is not possible until late in the procedure
Solution Approach 1:
The self-complementary sequence is incorporated into the primer before the MDA reaction begins, performing the sample identification function in advance. This preliminary action eliminates the need for late-stage barcode addition and enables immediate identification of samples throughout the procedure.
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
The method minimizes amplification bias, facilitates pooling of samples, and reduces sample preparation costs, improving sequencing efficiency and error correction in high-throughput nucleic acid sequencing.
Implementation Method 1
The self-complementary sequences may form a hairpin structure
Implementation Method 2
a DNA polymerase having a strand displacement activity
Implementation Method 3
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.