Fragmented Nucleic Acid Joining via Self-Complementary Internal Primers
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Solution Overview
Problem
Current methods for nucleic acid extraction from formalin-fixed and paraffin-embedded (FFPE) tissue and circulating cell-free DNA (cfDNA) face challenges due to fragmentation and low abundance, which complicates PCR-based cancer diagnosis, especially for mutations spread over multiple codons like EGFR exon 20.
Innovation Solution
A homogeneous method involving external and internal primers that anneal and extend to produce full-length target nucleic acid sequences from fragmented DNA, utilizing self-complementary internal primers to stitch together overlapping fragments, enabling exponential and homogeneous pre-enrichment for downstream PCR detection.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If conventional PCR methods are used on fragmented nucleic acids, then the detection process is simple, but the detection accuracy and sensitivity are insufficient for fragmented DNA
Solution Approach 1:
The method divides the amplification process into two distinct stages: first, external primers perform initial amplification on fragmented nucleic acids; second, internal primers perform stitching amplification to join fragments into full-length sequences. This segmentation allows each primer pair to specialize in a specific function, improving detection accuracy for fragmented DNA while maintaining manageable procedural complexity through clear stepwise execution.
2Productivity
If multiple discrete steps and reagents are used for target pre-enrichment, then the enrichment effectiveness is improved, but the process complexity and time consumption increase
Solution Approach 1:
The method merges target pre-enrichment and full-length sequence generation into a single unified PCR process. External primers and internal primers work cooperatively in one reaction system: external primers first amplify target regions from fragmented DNA, then internal primers stitch these amplification products into full-length sequences. This eliminates the need for separate pre-enrichment steps, reducing both process time and procedural complexity while maintaining high enrichment effectiveness.
Solution Approach 2:
The external primers perform preliminary amplification of target regions from fragmented nucleic acids before the internal primers perform the stitching operation. This preliminary action ensures that sufficient target material is available for the subsequent full-length reconstruction, improving overall enrichment effectiveness without requiring separate pre-enrichment steps.
3Productivity
If linear amplification methods are used, then the reagent requirements are reduced, but the enrichment efficiency is insufficient for low-abundance targets
Solution Approach 1:
The PCR-based method enables continuous exponential amplification throughout the reaction process. Both external and internal primers participate in cyclic amplification reactions that exponentially increase target sequence abundance. This continuous exponential action dramatically improves enrichment efficiency for low-abundance targets compared to linear methods, while the in-situ stitching approach minimizes additional reagent requirements by performing full-length reconstruction within the same reaction system.
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 joins fragmented nucleic acids to generate full-length targets, enhancing the sensitivity and accuracy of mutation detection in challenging samples like FFPE and cfDNA, overcoming the limitations of fragmented DNA sizes and low abundance.
Implementation Method 1
a pair of internal primers complementary to each other including a third nucleic acid sequence and a fourth nucleic acid sequence, the third nucleic acid sequence configured to hybridize to the first antisense strand of the first amplification product
Implementation Method 2
annealing a first 3' end region of the first sense strand with a second 3' end region of the first anti-sense strand, wherein the first 3' end region of the first sense strand primes the extension of the first sense strand over the first anti-sense strand
Data Source
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AI summary
Methods and kits for joining fragmented nucleic acid sequences together are provided, including performing an amplifying step including contacting a sample suspected of including a fragmented target nucleic acid with a pair of external primers and a pair of self- complementary internal primers, and generating a full length target nucleic acid. The methods can include performing an amplifying step, a hybridizing step, and a detecting step. Furthermore, kits are provided that are designed for the detection of a target nucleic acid sequence.