Mini-Monomer Cassette for Direct Promoter Identification
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Solution Overview
Problem
Current methods for identifying promoter sequences, RNA stability modifiers, and transcriptional modifying sequences are inefficient as they do not allow for the cloning or evaluation of cloned promoters and often require indirect identification through expressed sequence tags, lacking the ability to directly recover functional sequences from eukaryotic or random DNA sources.
Innovation Solution
A DNA plasmid construct featuring a mini-monomer cassette with ribozyme cleavage sites and a catalytic core, along with a multiple cloning site and primer annealing polynucleotides, allows for the transcription, self-cleavage, and circularization of RNA transcripts, enabling the direct identification and recovery of functional promoters and modifying sequences through reverse transcription and PCR.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If computer annotation of sequenced genomes is used to identify promoter sequences, then promoter sequences can be identified through bioinformatics analysis, but the method does not allow for cloning or evaluation of the identified promoters and cannot directly recover functional sequences
Solution Approach 1:
The method segments the identification process into two distinct phases: (1) in silico prediction using computer annotation to identify candidate promoter sequences, and (2) in vitro functional validation through cloning into reporter constructs. This segmentation allows each phase to optimize for its specific purpose while collectively achieving both accurate identification and functional verification.
Solution Approach 2:
The patent introduces an intermediary step between computational prediction and functional analysis: the cloning of predicted promoter sequences into reporter gene constructs. This intermediary allows the transition from in silico data to in vitro functional evaluation, enabling direct recovery and testing of candidate promoters while maintaining the efficiency of computational screening.
2Loss of information
If indirect identification through expressed sequence tags is used, then promoter regions can be located based on transcription start sites, but the method lacks the ability to directly recover and evaluate functional promoter sequences
Solution Approach 1:
The method merges two complementary approaches: (1) indirect identification through expressed sequence tags to locate promoter regions based on transcription start sites, and (2) direct recovery of functional sequences through cloning and reporter assays. This combination retains the advantages of both methods while overcoming their individual limitations.
Solution Approach 2:
The patent performs preliminary computational analysis and EST-based localization before conducting functional cloning experiments. This preliminary action narrows down the search space to high-probability candidate regions, making the subsequent direct recovery and functional evaluation more efficient and accurate.
3Manufacturing precision
If upstream sequence determination is required for promoter identification, then complete promoter context can be obtained, but the process becomes more complex and time-consuming
Solution Approach 1:
The method applies partial action by determining only the essential upstream sequences required for functional promoter activity rather than obtaining complete genomic context. Through systematic truncation and deletion analysis, the approach identifies the minimal functional elements needed, reducing time and complexity while maintaining identification accuracy.
Solution Approach 2:
The patent employs a dynamic, iterative approach to upstream sequence determination: starting with larger upstream regions and progressively truncating to identify minimal functional elements. This dynamic strategy adapts the sequencing effort to the actual functional requirements, avoiding unnecessary analysis of non-functional regions and reducing overall process time.
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 straightforward identification and recovery of functional promoters and modifying sequences, bypassing the need for upstream sequence determination and allowing for in vivo or in vitro expression, with deep sequencing analyzing the relative strength of recovered sequences.
Implementation Method 1
a mini-monomer cassette, comprising a first ribozyme cleavage site and a second ribozyme cleavage site, and comprising between the first ribozyme cleavage site and the second ribozyme cleavage site: (i) a ribozyme catalytic core
Implementation Method 2
reverse transcriptase polymerase chain reaction (RT-PCR) primer annealing polynucleotides
Data Source
AI summary
Provided are constructs and methods useful for the screening and identification of polynucleotide sequences of interest, in particular promoters, RNA stability modifying sequences and transcriptional modifying sequences.