Selective Transcript Extension Using Blocking Oligonucleotides
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Solution Overview
Problem
Existing methods for next-generation sequencing (NGS) suffer from PCR amplification bias, where high expresser genes dominate, skewing the native gene expression profile, and there is a need for selective amplification and extension of sequences of interest to accurately capture gene expression profiles in molecular biology.
Innovation Solution
A method involving the use of blocking oligonucleotides that specifically bind to undesirable nucleic acid species, combined with oligonucleotide probes, reduces the amplification and extension of these species, allowing for the selective extension of target nucleic acid molecules.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Quantity of substance
If PCR amplification is performed in excess cycles to adequately amplify low expresser genes, then the amplification of low expresser genes is improved, but the native gene expression profile is skewed by dominating high expresser PCR products
Solution Approach 1:
The patent extracts and removes high expresser genes (undesirable nucleic acid species) from the PCR reaction mixture using blocking oligonucleotides before amplification. This extraction of the problematic component allows subsequent PCR cycles to amplify low expresser genes without the dominating effect of high expresser products, resolving the contradiction between adequate amplification and profile accuracy.
Solution Approach 2:
The patent applies preliminary anti-action by introducing blocking oligonucleotides that specifically bind to high expresser genes before the PCR amplification process begins. This pre-blocking prevents these high expresser sequences from being amplified, thereby preventing the skewing of gene expression profiles that would otherwise occur during extended PCR cycling.
2Measurement precision
If Molecular Indexing is used to correct for PCR product bias, then the gene expression measurement is improved, but high expresser genes still dominate the sequencing run with little contribution to experimental interpretation
Solution Approach 1:
The patent removes high expresser genes from the sample before sequencing by using blocking oligonucleotides to prevent their amplification. This extraction ensures that these genes do not dominate the sequencing run, allowing sequencing resources to be productively allocated to low expresser genes that provide more valuable experimental information.
Solution Approach 2:
The patent performs the removal of high expresser genes as a preliminary action before the sequencing step. By blocking these genes during the amplification phase, the patent ensures that the sequencing run efficiently captures the expression profiles of low expresser genes without being overwhelmed by abundant high expresser transcripts.
3Measurement precision
If blocking oligonucleotides are used to prevent amplification of high expresser genes, then the representation of low expresser genes is improved, but the complexity of the amplification process increases
Solution Approach 1:
The patent introduces blocking oligonucleotides as intermediary molecules that mediate the selective prevention of high expresser gene amplification. These oligonucleotides act as intermediaries between the PCR reagents and the target genes, specifically binding to high expresser sequences and preventing primer annealing, thereby improving low expresser gene representation through a controlled mechanism.
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 significantly reduces the amplification and extension of undesirable nucleic acid species, such as ribosomal and mitochondrial mRNAs, improving the accuracy of gene expression analysis by minimizing bias and enhancing the representation of low expresser genes.
Implementation Method 1
the blocking oligonucleotide specifically binds to at least one of the one or more undesirable nucleic acid species
Implementation Method 2
each of the plurality of oligonucleotide probes comprises a molecular label sequence and a target binding region capable of hybridizing to the plurality of nucleic acid target molecules
Data Source
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AI summary
Disclosed herein include methods and compositions for selectively amplifying and/or extending nucleic acid target molecules in a sample. The methods and compositions can, for example, reduce the amplification and/or extension of undesirable nucleic acid species in the sample, and/or allow selective removal of undesirable nucleic acid species in the sample.