Parallel Multiple Displacement Amplification With Lossless Barcoding
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Solution Overview
Problem
Current whole genome amplification technologies, such as MDA, face challenges in increasing parallelism and throughput, leading to high costs and inefficiencies in analyzing large numbers of cells, particularly for single-cell genome analysis at the single base sequence level, due to amplification bias and the difficulty in inserting barcodes without loss during the MDA process.
Innovation Solution
A nucleic acid amplification method using a first primer with a fixed second base sequence to distinguish the sample and a second primer that hybridizes to the target nucleic acid, enabling parallel multiple displacement amplification (bMDA) to increase parallelism and throughput, allowing for efficient library preparation and sequencing at reduced costs.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If traditional MDA is used to analyze single cells, then genome coverage and amplification fidelity are improved, but the cost and time increase linearly with the number of cells analyzed
Solution Approach 1:
The invention divides the amplification process into two stages: a first amplification stage using MDA with phi29 polymerase to generate amplified DNA, and a second amplification stage using barcode-containing primers to selectively amplify only the desired portions. This segmentation allows parallel processing of multiple samples while maintaining high genome coverage and fidelity in the first stage, then efficiently filtering and amplifying specific regions in the second stage.
Solution Approach 2:
The invention extracts and removes unwanted amplified DNA portions through selective primer binding in the second amplification stage. By using barcode-containing primers that specifically bind to desired regions, the method extracts only the relevant information while discarding redundant or unwanted amplification products from the first stage, thereby reducing cost and time for analyzing multiple cells.
2Productivity
If barcodes are inserted during MDA to enable parallel analysis, then throughput is improved, but amplification bias increases and barcode insertion accuracy decreases
Solution Approach 1:
The invention performs preliminary amplification of the entire genome using MDA with phi29 polymerase before introducing barcode-containing primers. This preliminary action ensures that the complete genome is amplified with high fidelity and uniformity first, and only then are the barcode-containing primers introduced to selectively amplify specific regions. This sequence prevents amplification bias because the barcode primers act on already-uniformly-amplified DNA rather than competing with random hexamer primers during the initial amplification.
Solution Approach 2:
The invention extracts the barcode-containing primers from the pool of all possible primers and uses them selectively in the second amplification stage. By separating the function of whole-genome amplification (performed by random hexamers in stage 1) from the function of selective amplification (performed by barcode-containing primers in stage 2), the method eliminates the amplification bias that would occur if all primers competed simultaneously. This extraction of the barcode primer function to a separate stage ensures accurate barcode insertion without compromising amplification uniformity.
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 enables high-throughput, cost-effective analysis of single-cell genomes at the single base sequence level by reducing library preparation time and costs, and improving amplification quality through lossless barcode insertion and efficient nucleic acid amplification.
Implementation Method 1
a first primer, a second primer and a nucleic acid polymerase to a sample containing a target nucleic acid, wherein the first primer is a polynucleotide consisting of a base sequence including a first base sequence that hybridizes to the target nucleic acid
Implementation Method 2
amplifying nucleic acids by adding a first primer, a second primer and a nucleic acid polymerase to a sample containing a target nucleic acid
Data Source
AI summary
One aspect relates to a nucleic acid amplification method that comprises the step of amplifying nucleic acids by adding a first primer, a second primer and a nucleic acid polymerase to a sample containing a target nucleic acid. According to the nucleic acid amplification method according to one aspect, by performing parallel multiple displacement amplification using the first primer containing the barcode sequence, the increased parallelism and throughput of MDA dramatically reduces the time and cost for NGS library production, the barcode bias phenomenon can be resolved by adjusting the ratio at which the first primer is added, and the quality of amplification can also be improved using a laser-based cell separation method. Therefore, the nucleic acid amplification method can be usefully used for large-scale single-cell whole genome analysis.