PCR Detection of Short Nucleic Acid Fragments via Template Extension
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Solution Overview
Problem
Traditional PCR methods fail to detect short nucleic acid fragments in urine samples due to degradation by DNases, as these fragments are often too short to contain both primer regions necessary for amplification.
Innovation Solution
A method involving the use of a nucleic acid template that partially hybridizes with the fragment, allowing extension by a polymerase and subsequent removal of the template, enabling the amplification of fragments that can be detected by PCR, even if they are initially too short.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If traditional PCR methods are used to detect nucleic acid fragments in urine samples, then the detection process is simple and direct, but short fragments are degraded by DNases and become too short to contain both primer regions necessary for amplification
Solution Approach 1:
The method performs preliminary actions by adding a nucleic acid template and polymerase before PCR amplification to extend short fragments in situ, converting them into full-length amplicons that contain both primer regions. This preliminary extension step ensures that even fragmented nucleic acids can be successfully amplified and detected by subsequent PCR.
Solution Approach 2:
The patent introduces a nucleic acid template as an intermediary that hybridizes to the short fragment and serves as a scaffold for polymerase-mediated extension. This intermediary template enables the conversion of non-amplifiable short fragments into amplifiable full-length products without requiring the fragments to be intact originally.
2Productivity
If the nucleic acid template is retained in the product after extension, then the extension reaction can be maintained, but the template may interfere with subsequent PCR amplification and detection
Solution Approach 1:
The method extracts or removes the nucleic acid template from the reaction product after the extension step is complete. This removal eliminates potential interference from the template during subsequent PCR amplification, ensuring that only the extended fragments are amplified and detected, thereby improving detection specificity.
Solution Approach 2:
The template is discarded after serving its purpose of enabling fragment extension. The useful information (extended fragment sequence) is recovered and preserved in the extended product, while the template itself is removed to prevent interference with downstream applications.
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 allows for the detection of previously undetectable nucleic acid fragments by reconstructing full-length amplicons, enhancing the sensitivity and specificity of PCR-based diagnostics, particularly for biomarkers like TB in urine samples.
Implementation Method 1
a nucleic acid template that partially hybridizes to and serves as an amplification template for a fragment of a nucleic acid target
Implementation Method 2
contacting the product of step (b) with (i) a second polymerase, (ii) a nucleotide triphosphate mixture, and (iii) a forward primer under conditions supporting polymerization of a nucleic acid strand complementary to the extended fragment
Data Source
AI summary
The present disclosure is directed to methods of detecting small fragments of known nucleic acid biomarkers.