Assembler Sequence for Fragmented Nucleic Acid Amplification
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Solution Overview
Problem
Existing molecular diagnostic assays face challenges in detecting short, degraded nucleic acid fragments, which are common in samples like urine and FFPE, due to the requirement for longer target sequences to accommodate primers and probes, making it difficult to detect single nucleotide variations accurately.
Innovation Solution
The method involves using an assembler sequence complementary to the target nucleic acid to extend short nucleic acid fragments, allowing for linear amplification and detection of genetic variations by annealing and extending the fragments with primers, thereby producing larger amplicons for further manipulation and analysis.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If standard molecular diagnostic assays are used to detect target nucleic acids, then detection of single nucleotide variations can be achieved, but the method fails when target nucleic acids are heavily degraded and shorter than the assay footprint
Solution Approach 1:
The assay is divided into two distinct phases: (1) a footprint reduction step that removes primers and probes to generate shorter target fragments, and (2) a detection phase that uses these reduced-footprint fragments. This segmentation allows the assay to work with degraded samples while maintaining single nucleotide variation detection capability.
Solution Approach 2:
The footprint reduction step is performed as a preliminary action before the actual detection. By pre-processing the target nucleic acids to reduce their footprint and remove interfering elements, the assay becomes adaptable to degraded samples while preserving the ability to detect single nucleotide variations in the subsequent detection phase.
2Measurement precision
If the target nucleic acid length is increased to accommodate primers and probes for specific detection, then single nucleotide variation detection is enabled, but the assay cannot detect short fragmented targets
Solution Approach 1:
Instead of requiring the target to be long enough to accommodate primers and probes (conventional approach), the invention inverts the approach by first removing primers and probes from the assay footprint, then performing detection with the remaining short target fragment. This inversion allows detection of single nucleotide variations without requiring long target sequences.
Solution Approach 2:
The invention extracts and removes the primer and probe components from the assay footprint, isolating only the essential target nucleic acid region needed for detection. This extraction eliminates the length requirement while preserving single nucleotide variation detection capability.
3Reliability
If PCR amplification is used to amplify target nucleic acids, then detection sensitivity is improved, but the method requires targets longer than the primer footprint which excludes degraded samples
Solution Approach 1:
The detection process is segmented into footprint reduction and detection phases, allowing amplified products from degraded samples to be detected without requiring the original target to meet minimum length requirements. This segmentation enables PCR amplification of short fragments followed by effective detection.
Solution Approach 2:
Footprint reduction is performed as a preliminary step before detection, enabling the use of PCR amplification on degraded samples followed by detection of the amplified products. This preliminary action removes the length barrier that would otherwise prevent PCR-based detection of degraded nucleic acids.
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 enables the efficient assembly and amplification of short nucleic acid fragments, preserving single nucleotide variations and allowing for accurate detection of genetic variants, even in highly fragmented samples, by producing longer amplicons that can be interrogated effectively.
Implementation Method 1
annealing said short nucleic acid fragments to said assembler sequence
Implementation Method 2
said assembler sequence is substantially complementary to said target nucleic acid
Implementation Method 3
extending said short nucleic acid fragments in the 3'-direction by polymerase
Data Source
Figure 1
Figure 1
AI summary
The present invention provides methods of amplifying a fragmented target nucleic acid containing short target nucleic acid fragments utilizing an assembler sequence to convert these short fragments into longer sequences enabling their identification and interrogation. This is particularly important when attempting to identify small genetic variations, such as SNVs, present in highly fragmented nucleic acid samples. Amplification is accomplished by hybridizing the short target nucleic acid sequences to the assembler sequence, where these short sequences serve as primers for extension. Since the fragmented target nucleic acids that contain SNVs are utilized as primers on the assembler sequence they are preserved during amplification and can be detected.