Microsatellite Length Measurement After Partial Mutagenesis
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Solution Overview
Problem
Accurate measurement of microsatellite length variations (MSLV) is challenging due to their instability during amplification and sequencing, particularly in cancerous tissues, leading to high error rates and inaccurate quantitation.
Innovation Solution
A method involving partial mutagenesis of nucleic acid templates to disrupt microsatellites, followed by sequencing and selecting reads with specific disruption criteria to measure microsatellite lengths accurately.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If microsatellites are sequenced directly to measure length variations, then the measurement process is simple, but the error rate is high due to polymerase slippage and amplification instability
Solution Approach 1:
The patent applies preliminary action by performing partial mutagenesis on the microsatellite region before sequencing. This pre-treatment disrupts the repetitive structure that causes polymerase slippage during amplification, thereby stabilizing the microsatellite for accurate sequencing while preserving the flanking regions for length measurement.
Solution Approach 2:
The patent applies local quality by selectively mutagenizing only the microsatellite repeat region while leaving the flanking sequences intact. This localized mutagenesis disrupts the problematic repetitive structure without affecting the stable flanking regions, enabling accurate measurement of microsatellite length variations.
2Measurement precision
If complete mutagenesis is performed to stabilize microsatellites, then measurement accuracy improves, but the microsatellite structure is completely destroyed making length measurement impossible
Solution Approach 1:
The patent applies partial action by performing partial mutagenesis that disrupts the microsatellite structure sufficiently to prevent polymerase slippage and stabilize length measurement, but not so extensively as to completely destroy the microsatellite. The flanking regions remain intact, allowing accurate measurement of the original microsatellite length.
3Productivity
If high throughput sequencing platforms are used to sequence microsatellites, then processing speed increases, but reading accuracy for mononucleotide tracts decreases
Solution Approach 1:
The patent applies preliminary action by performing partial mutagenesis before sequencing. This pre-treatment modifies the microsatellite structure to reduce polymerase slippage and improve sequencing accuracy, thereby enabling high throughput platforms to read mononucleotide tracts accurately while maintaining high processing speed.
Data Source
AI summary
This invention provides a method for obtaining microsatellite lengths from initial nucleic acid templates each of which comprise a microsatellite and two flanking portions, which define the microsatellite and its locus.