Isoform Detection via Segmented PCR Primers
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Solution Overview
Problem
Current understanding of human somatostatin receptor type 5 (sst5) lacks description of alternative isoforms, limiting the ability to study their role in various pathologies and develop targeted therapies.
Innovation Solution
Identification and cloning of two new isoforms, sst5B and sst5C, with five and four transmembrane domains respectively, using RACE-PCR technique, allowing for the production of recombinant DNA molecules and polypeptides for studying their function and modulation in response to drugs.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If conventional PCR detection methods are used for sst5, then only the known sst5A isoform can be detected, but alternative isoforms (sst5B and sst5C) remain undetected
Solution Approach 1:
The invention divides the detection approach into separate PCR assays for each isoform (sst5A, sst5B, sst5C) with specific primers tailored to each variant's unique sequence regions. This segmentation allows each primer set to specifically amplify its target isoform without cross-reactivity, enabling precise detection of individual isoforms that were previously indistinguishable by conventional methods.
2Loss of information
If RACE-PCR technique is used to clone new isoforms, then novel isoforms can be identified, but the process requires complex experimental procedures
Solution Approach 1:
The invention performs preliminary RACE-PCR cloning experiments to identify and characterize new isoforms before developing the diagnostic PCR assays. By conducting the complex cloning and sequencing work upfront to obtain complete cDNA sequences of sst5B and sst5C, the invention prepares all necessary sequence information in advance, which then simplifies the design of specific primers for routine detection without requiring repeated complex cloning procedures.
3Measurement precision
If selective primers are designed for each isoform, then specific detection is achieved, but primer design becomes more complex
Solution Approach 1:
The invention applies local quality by designing primers that target specific unique regions of each isoform's sequence. The sst5A primers target regions specific to the canonical isoform, while sst5B and sst5C primers target their respective unique sequences. This localized targeting of specific sequence regions ensures high specificity for each isoform detection while maintaining a relatively simple overall assay design.
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
Enables the development of selective drugs and tools to study and modulate the new isoforms, facilitating the understanding of their role in diseases and potential therapeutic applications.
Implementation Method 1
cloning of the porcine sst5 through the RACE PCR technique
Implementation Method 2
partial, and then total sequences of messenger RNA variants had been obtained through alternative adjustment
Data Source
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AI summary
The present invention concerns two human nucleic acids comprising sequences which code two novel isoforms of human somatostatin receptor type 5 produced by alternative adjustment, called sst5B and sst5C and with possible uses in tumoral processes. Additionally, the invention concerns oligonucleotide pairs used for the differential detection of said isoforms by means of the PCR technique in different tissues.