Iterative Primer Extension for Nucleic Acid Variant Isolation
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Solution Overview
Problem
Current methods for determining nucleic acid sequences and sequence differences are not efficient for rapid and accurate discovery of genetic or epigenetic variations, which is crucial for biomedical research and medical applications.
Innovation Solution
An iterative base-by-base primer extension method is employed to isolate nucleic acids with sequences different from a reference sequence by annealing a polymerization primer and using a polymerization reagent pool with nucleotide polymerase and specific nucleotide bases to identify and isolate variant nucleic acids.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If current sequencing methods are used, then nucleic acid sequences can be determined, but the process is not efficient for rapid and accurate discovery of genetic or epigenetic variations
Solution Approach 1:
The method segments the nucleic acid analysis process into discrete base-by-base extension steps, where each position in the locus is interrogated individually through sequential primer extension reactions. This segmentation allows for systematic comparison against the reference sequence at each position, enabling rapid identification of variations while maintaining accuracy through stepwise verification.
Solution Approach 2:
The method employs preliminary action by pre-annealing polymerization primers to the nucleic acid sample before the extension reaction. The primers are designed to bind immediately upstream of the locus of interest, establishing a fixed starting point for the iterative extension process. This preliminary positioning enables efficient and accurate traversal through each base position to detect variations.
2Productivity
If an iterative base-by-base primer extension method is employed, then variant nucleic acids can be isolated efficiently, but the process complexity increases
Solution Approach 1:
The method employs a universal polymerization primer design that can be applied to any nucleic acid sample containing the locus of interest. The same primer annealing and extension protocol is used regardless of the specific sample source or variation type, making the method universally applicable while maintaining efficiency. The polymerization reagent pool also serves multiple functions by providing all necessary nucleotides and polymerase activity in a single reagent system.
Solution Approach 2:
The method uses the polymerization primer as an intermediary element that mediates between the nucleic acid template and the detection system. The primer serves as a fixed reference point from which the iterative extension proceeds, allowing systematic interrogation of each base position. This intermediary approach simplifies the overall process by providing a consistent starting point and mechanism for variation detection across different samples.
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 allows for the efficient isolation and identification of nucleic acid molecules with sequence differences, enabling improved analytical assays and genetic measurements in various fields.
Implementation Method 1
annealing a polymerization primer to the nucleic acid molecules immediately upstream of the locus of interest
Implementation Method 2
contacting the polymerization primer-annealed nucleic acid molecules with a polymerization reagent pool under nucleic acid polymerization conditions, wherein the polymerization reagent pool comprises a nucleotide polymerase
Implementation Method 3
the two or more nucleotide bases are selected based on the identity of the next two different nucleotide bases predicted to be incorporated at unoccupied sites downstream of the polymerization primer
Data Source
AI summary
The invention is drawn to isolating sequence variants of a genetic locus of interest using a modified iterative primer extension method. The nucleic acids analyzed are generally single stranded and have a reference sequence which is used as a basis for performing iterative single nucleotide extension reactions from a hybridized polymerization primer. The iterative polymerization reactions are configured such that polymerization of the strand will continue if the sequence of the nucleic acid being analyzed matches the reference sequence, whereas polymerization will be terminated if the nucleic acid being analyzed does not match the reference sequence. Nucleic acid strands that have mutations can be isolated using a variety of methods and sequenced to determine the precise identity of the mutation/polymorphism. By performing the method on both strands of the nucleic acid being analyzed, virtually all possible mutations can be identified.