Loopable Split Primers for Multiplex PCR Barcode Accuracy
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Solution Overview
Problem
Existing molecular barcode methods in next-generation sequencing face challenges in suppressing primer dimer formation, avoiding barcode resampling, and reducing nonspecific primer binding, which are crucial for accurate detection of genetic variations.
Innovation Solution
The use of split primers with a molecular indexing section positioned between target-specific sections and an adaptor section, along with stem-forming sections to form loop structures, which protect universal adaptor sequences and molecular indexing sequences, thereby reducing primer dimers and non-specific binding.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If molecular barcodes or indexing sequences are used in highly multiplex PCR, then quantitative bias is reduced and PCR artifacts can be identified, but primer dimer formation increases and nonspecific primer binding occurs
Solution Approach 1:
The primer is divided into multiple distinct sections: a target-specific section for binding to the template DNA, an adaptor section containing the molecular barcode, and a blocking section with a sequence complementary to the target-specific section. This segmentation allows each part to perform its specific function while preventing unwanted interactions between primers.
Solution Approach 2:
The blocking section is designed to preemptively prevent primer dimer formation by hybridizing to the target-specific section of the same primer or complementary primers. This preliminary anti-action occurs before amplification can proceed, stopping harmful primer-primer interactions in advance.
2Reliability
If molecular barcodes or indexing sequences are used in highly multiplex PCR, then PCR artifacts can be identified and separated from real variants, but barcode resampling occurs and amplification accuracy decreases
Solution Approach 1:
The blocking section prevents barcode resampling and primer concatamer formation by hybridizing to the target-specific section, thereby protecting the integrity of the molecular barcode system and ensuring accurate amplification of the intended target sequences.
Solution Approach 2:
The blocking section acts as an intermediary element that mediates between the target-specific section and the adaptor section. It controls the conformation and interaction of the primer, ensuring that the molecular barcode functions correctly without causing harmful side effects.
3Ease of manufacture
If traditional primers are used in highly multiplex PCR, then primer design is simpler, but primer concatamers form and amplification efficiency decreases
Solution Approach 1:
The primer is divided into multiple distinct sections: a target-specific section for binding to the template DNA, an adaptor section containing the molecular barcode, and a blocking section with a sequence complementary to the target-specific section. This segmentation allows each part to perform its specific function while preventing unwanted interactions between primers.
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 enhances assay specificity and accuracy in multiplex PCR, enabling effective molecular counting and detection of genetic variations, particularly in highly multiplex reactions.
Implementation Method 1
the stem-forming section is hybridizable to the 3'-portion of the target-specific section to form a stem structure and a loop comprising the adaptor section and the molecular indexing section
Data Source
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AI summary
Disclosed here is a composition comprising a primer that is (a) a loopable primer comprising a target-specific section, an adaptor section, and a stem-forming section, wherein the stem-forming section is hybridizable to a portion of the target-specific section to form a stem structure, or (b) a split primer comprising a first target-specific section, a second target-specific section, and an adaptor section positioned between the first target-specific section and the second target-specific section, or (c) a split-loopable primer comprising a first target-specific section, a second target-specific section, a stem-forming section positioned between the first target-specific section and the second target-specific section, and an adaptor section, or comprising a first adaptor section, a second adaptor section, a stem-forming section positioned between the first adaptor section and the second adaptor section, and a target-specific section. Also disclosed is a method for amplifying a target locus of interest from a template DNA, comprising at least two pre-amplification cycles using the loopable primer, the split primer and/or the split-loopable primer, wherein each amplification cycle comprises annealing the primer to the template DNA or pre-amplification product thereof and elongating the annealed primer. Further disclosed is a kit for amplifying a target locus of interest, comprising the loopable primer, the split primer, and/or the split-loopable primer.