Barcoded MDA Primers With Complementary Blocking Against Self-Priming
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Solution Overview
Problem
Existing multiple displacement amplification (MDA) techniques using barcoded randomized primers face issues of undesired self-priming due to longer primers using each other as templates, leading to significant side-products.
Innovation Solution
The use of barcoded randomized primers with a complementary sequence, either covalently linked as a hairpin or separate, to block self-priming, combined with partitioning and sequencing technologies to maintain unique molecular identifiers.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If barcoded randomized primers are used for MDA, then amplification coverage and tracking capability are improved, but self-priming side-products increase
Solution Approach 1:
The patent extracts and removes the harmful self-priming capability by designing the complementary sequence to be non-complementary to the barcode and random sequence portions, effectively taking out the harmful self-annealing property while retaining the beneficial tracking capability
Solution Approach 2:
The complementary sequence acts as an intermediary element that blocks self-priming by annealing to the primer in a controlled manner, mediating between the primer's amplification function and the need to prevent self-priming side reactions
2Loss of information
If longer primers with barcodes are used, then molecular identification capability is improved, but self-priming occurs more frequently
Solution Approach 1:
The patent segments the primer into distinct functional regions (barcode sequence, random sequence, and complementary sequence), allowing each segment to perform its specific function while preventing unwanted interactions between them
Solution Approach 2:
The patent applies local quality by making different portions of the primer have different properties: the barcode portion maintains identification capability, the random sequence provides annealing specificity, and the complementary sequence locally blocks self-priming through controlled annealing
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
Reduces background of undesired self-priming products, ensuring desired MDA products with improved accuracy and efficiency.
Implementation Method 1
contacting the plurality of oligonucleotides to sample DNA under conditions in which the complementary nucleic acid anneals to an oligonucleotide and allows for annealing of the 3' random sequence to the DNA
Implementation Method 2
extending the 3' random sequence in a template-dependent manner with a strand-displacing polymerase to generate extended oligonucleotides comprising a 3' sequence complementary to the DNA
Data Source
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AI summary
Improved multiple displacement amplification (MDA) reagents and methods are provided.