Multiplex PCR Primer Design for Gene Fusion Detection
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Solution Overview
Problem
Current methods for detecting gene fusions associated with cancers, such as EML4-ALK in non-small cell lung cancer, are invasive, require large biopsy samples, and have high false positive rates due to limitations in biopsy processing and the variable nature of gene fusions, leading to a low testing rate and missed opportunities for effective treatments.
Innovation Solution
A novel multiplex method using specific primer pairs that amplify across fusion sites and regions 5' and 3' of the fusion site, along with control primers, to detect genetic fusions in small biological samples like circulating free RNA, allowing for non-invasive testing and accurate identification of gene fusions.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If biopsy of tumor tissue is performed for gene fusion detection, then detection can be carried out by FISH or IHC, but the procedure is invasive and requires large biopsy samples from weakened patients
Solution Approach 1:
The invention extracts the essential detection function from invasive tissue biopsy and transfers it to non-invasive liquid biopsy methods. By designing primers that specifically amplify fusion transcripts in circulating tumor DNA/RNA from blood samples, the method achieves reliable detection without requiring surgical tissue extraction, thus resolving the contradiction between detection reliability and procedural invasiveness
Solution Approach 2:
The invention introduces circulating tumor DNA/RNA as an intermediary substance that carries genetic fusion information from the tumor to the blood circulation. This intermediary allows detection of gene fusions through simple blood draws rather than direct tissue biopsy, eliminating the need for invasive procedures while maintaining detection accuracy through molecular amplification of the intermediary carrier
2Measurement precision
If multiple tissue sections are observed by skilled cytologists to reduce false positives, then detection accuracy improves, but the testing becomes time and resource intensive with low testing rate
Solution Approach 1:
The invention replaces the mechanical and manual process of visual inspection by skilled cytologists with an automated molecular biology system. The PCR amplification and detection methods automatically identify fusion transcripts with high specificity, eliminating the need for multiple manual tissue section reviews while dramatically increasing testing throughput and productivity
Solution Approach 2:
The invention performs preliminary enrichment of fusion-specific genetic material through targeted PCR amplification before detection. By pre-amplifying only the relevant fusion transcripts using specifically designed primers, the method concentrates the signal of interest and eliminates the need for extensive manual review of multiple tissue sections, thereby improving both accuracy and testing efficiency
3Difficulty of detecting and measuring
If RT-PCR is used to detect gene fusions, then detection can be performed, but the highly variable nature of gene fusions and limited quality of genetic material from FFPE tissue results in unsuccessful detection
Solution Approach 1:
The invention applies local quality by designing primers with specific characteristics optimized for different regions of fusion transcripts. The primer pairs are carefully designed to target conserved regions flanking variable fusion sites, with specific annealing temperatures and lengths optimized for the local sequence context. This localized optimization ensures reliable amplification across the highly variable landscape of different gene fusions while accounting for the degraded nature of FFPE-derived genetic material
Solution Approach 2:
The invention employs parameter changes by optimizing PCR conditions including annealing temperature, magnesium concentration, and cycle parameters to accommodate the degraded and variable nature of FFPE genetic material. The method adjusts amplification parameters to enhance sensitivity for low-quality templates while maintaining specificity, thereby overcoming the limitations of RT-PCR with FFPE samples and achieving reliable detection across diverse fusion variants
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 method enables sensitive and specific detection of gene fusions in minimal sample quantities, reducing the need for invasive biopsies and improving the detection rate, thereby facilitating timely and effective treatment recommendations for cancer patients.
Implementation Method 1
amplify across fusion sites and regions 5' and 3' of the fusion site
Data Source
AI summary
Provided herein are methods and compositions for detecting gene fusions, e.g., relevant to cancer. The present methods and compositions can be used to detect gene fusions with very high sensitivity and specificity. The present methods and compositions can detect gene fusions, e.g., in free circulating tumor RNA from a plasma sample.