Universal Inosine Template for Random Allelic Series Construction
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Solution Overview
Problem
Current methods for large-scale mutagenesis of specific DNA sequences are limited by the need to synthesize numerous oligonucleotides, making it costly and inefficient to produce allelic series with single nucleotide changes, which is essential for understanding protein function and variant interpretation in humans.
Innovation Solution
A method involving linear PCR with universal base triphosphates containing a blocking group, followed by removal of the blocking group and exponential PCR, generates a mutational library with single point mutations evenly distributed throughout the target nucleic acid, eliminating the need for multiple oligonucleotides and reducing costs.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If oligonucleotides are synthesized en masse to be used as templates for mutagenesis, then allelic series can be constructed, but the cost and complexity increase significantly
Solution Approach 1:
The patent uses a universal base (inosine) that can pair with all four natural bases (A, T, C, G), allowing a single oligonucleotide template to generate all possible single nucleotide mutations at a target position. This eliminates the need for four separate oligonucleotides for each mutation site, reducing the number of components from hundreds to thousands down to a minimal set.
Solution Approach 2:
The patent introduces an intermediary universal base (inosine) that mediates between the oligonucleotide template and the target DNA. This intermediary allows non-specific base pairing during initial annealing, enabling a single template to guide the introduction of multiple different nucleotides through subsequent PCR amplification with normal dNTPs.
2Productivity
If traditional mutagenesis methods are used, then allelic series can be produced, but the production time and cost increase
Solution Approach 1:
A single universal oligonucleotide template containing inosine at the desired mutation position can generate all four possible single nucleotide variants (A, T, C, G) at that position through PCR amplification. This universal template replaces the need to purchase and handle hundreds of individually synthesized oligonucleotides, dramatically reducing both quantity required and production time.
Solution Approach 2:
The patent performs preliminary annealing of the universal oligonucleotide template to the target DNA before PCR amplification. This preliminary step allows the universal base to establish initial binding, after which standard PCR with normal dNTPs efficiently generates the complete allelic series, separating the template preparation from the mutation generation steps.
3Manufacturing precision
If linear PCR with blocking group is used, then single point mutations are generated, but additional processing steps are required
Solution Approach 1:
The patent extracts the blocking group from the universal base triphosphate before incorporating it into the oligonucleotide template. This removal of the blocking group allows the universal base to be incorporated during linear PCR without preventing subsequent exponential amplification, thereby reducing the number of additional processing steps needed while maintaining single point mutation precision.
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 rapid and cost-effective production of allelic series with single nucleotide changes, improving the interpretation of genetic variants and protein function analysis, while minimizing secondary mutations and size restrictions.
Implementation Method 1
creating a reaction mixture comprising the target nucleic acid, polymerase, a forward primer, deoxynucleotide triphosphates (dNTPs) and universal base triphosphates comprising a blocking group; performing linear PCR, wherein the linear PCR generates products of various lengths, wherein each product comprises about 1 universal base comprising a blocking group, and wherein the linear PCR stops after incorporating the universal base triphosphate comprising a blocking group
Implementation Method 2
dephosphorylating 2′-deoxy, 3′-O—NH2 adenosine triphosphate with a phosphatase
Implementation Method 3
deaminating the product from (a) with a deaminase
Implementation Method 4
phosphorylating the product from (b) with three different kinases
Data Source
AI summary
The present disclosure provides a method of making a systematic single point mutation in a target nucleic acid and a method of generating a mutational library comprising target nucleic acids with single point mutations. The mutational library comprises target nucleic acids with single point mutations distributed evenly throughout the target nucleic acid.