Stem-Loop Primer Design for Multiplex PCR Dimer Reduction
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
In multiplex PCR, the formation of primer dimers due to complementary regions at the 3' ends of primers leads to inefficient amplification of desired DNA sequences, especially at high primer concentrations, which is challenging to address with existing methods that require modified primers and additional enzymes, increasing costs and complexity.
Innovation Solution
The method involves using a first forward primer and a second reverse primer with a complementary region at their 3' ends, where a partial or full 5' sequence of the first forward primer is incorporated between universal tags to form a stem-loop structure that inhibits further primer dimer amplification by increasing the annealing temperature in subsequent PCR cycles.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Loss of information
If multiple primer sets are used in multiplex PCR to target multiple genes, then additional information can be gained from a single test run, but the accumulation of primer-dimers increases and decreases assay sensitivity
Solution Approach 1:
The invention converts the harmful complementary region at the 3' end of primers (which causes primer-dimer formation) into a beneficial stem-loop structure by incorporating a partial or full 5' sequence of the forward primer between universal tags. This stem-loop structure forms on the primer-dimer and prevents further amplification, thereby converting the harmful primer-dimer into a non-amplifiable structure that does not compete with target sequences for reagents.
2Productivity
If primer concentrations are increased to improve amplification efficiency, then more primers are available for target amplification, but the chances of primer-dimer formation increase significantly
Solution Approach 1:
The invention applies preliminary anti-action by designing primers with incorporated 5' sequences that pre-form stem-loop structures. These stem-loops are prepared in advance and will form on any primer-dimers that occur during PCR, preventing their further amplification. This preliminary structural design counteracts the harmful effect of high primer concentrations before primer-dimers can consume significant reagents.
3Object-generated harmful factors
If HANDS PCR method is used with common tail sequences to reduce primer-dimer amplification, then stem-loop structures inhibit primer-dimer formation, but the targeted amplicons must be long enough to minimize inhibitory effects, which limits applicability
Solution Approach 1:
The invention applies local quality by making the stem-loop structure local to the primer-dimer itself rather than requiring global changes to all primers with common tails. By incorporating the partial or full 5' sequence of the forward primer between universal tags, the stem-loop forms specifically on the primer-dimer structure where it is needed, without requiring all primers to have the same tail sequence or requiring amplicons to be of minimum length.
4Object-generated harmful factors
If modified bases and additional enzymes are used to activate primers upon specific hybridization, then primer-dimer formation is reduced, but the cost and complexity of the method increase
Solution Approach 1:
The invention applies self-service by designing primers that automatically form stem-loop structures on their own without requiring external enzymes or modified bases for activation. The incorporated 5' sequences within the universal tags enable the primers to self-regulate and prevent primer-dimer amplification through intrinsic structural formation, eliminating the need for additional enzymatic steps or modified nucleotides.
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 effectively reduces primer dimer formation, allowing for the preferential amplification of target sequences by blocking further annealing of primers to primer dimer strands, thereby improving the sensitivity and specificity of multiplex PCR, especially in high multiplex levels like next-generation sequencing applications.
Implementation Method 1
A PD consists of primer molecules that hybridize to each other due to strings of complementary bases, particularly at the 3'-ends of the primers
Implementation Method 2
the annealing temperature is elevated for the subsequent amplification cycles which are driven entirely by the tail-specific primer. Consequently, the single strands from all PCR products, including desired amplicons and side-products such as PD, have complementary 5' and 3' ends leading to the formation of the same stem-loop structures
Data Source
Figure 1A
Figure 1B
Figure 2
AI summary
The present invention reduces primer-dimer amplification in a multiplex polymerase chain reaction (PCR). When a first forward primer (F1) and a second reverse primer (R2) have a complementary region at their 3'ends, primer dimers may form. The present method uses a primer comprising a 5'-end partial sequence or a full sequence of a first forward primer (F1^) in between a first tag (t1) and R2 to reduce the primer-dimer (F1_R2) amplification.