Duplex-Specific Nuclease DNA Probe Preparation
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Solution Overview
Problem
Existing methods for generating DNA probes are hindered by the presence of repetitive sequences, which reduce specificity and require costly blocking nucleic acids or complex processes, making it difficult to produce high-specificity probes for applications like FISH and CGH without distorting genomic hybridization patterns.
Innovation Solution
The use of duplex-specific nucleases (DSN) to selectively cleave and remove repetitive sequences from DNA probes, allowing for the production of repeat-depleted probes that eliminate the need for blocking DNA, thereby enhancing probe specificity and reducing costs and complexity.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If blocking nucleic acids are used to inhibit hybridization of repetitive sequences, then probe specificity is improved, but cost increases and genomic hybridization patterns are distorted
Solution Approach 1:
The patent extracts and removes repetitive sequences from the probe preparation process by using duplex-specific nucleases to selectively digest double-stranded repetitive DNA during probe amplification. This eliminates the need for blocking nucleic acids and prevents distortion of genomic hybridization patterns, as the repetitive sequences are physically removed rather than blocked.
2Manufacturing precision
If hydroxyapatite chromatography is used to remove repetitive sequences, then probe specificity is improved, but the process becomes cumbersome and requires strict optimization
Solution Approach 1:
The patent replaces the mechanical chromatography-based hydroxyapatite removal system with an enzymatic system using duplex-specific nucleases. This enzymatic approach selectively digests repetitive sequences during PCR amplification without requiring complex chromatography equipment or strict optimization of physical conditions, significantly simplifying the manufacturing process.
3Manufacturing precision
If PCR assisted affinity chromatography is used to remove repeats, then probe specificity is improved, but the process becomes complex and time-consuming
Solution Approach 1:
The patent merges the repeat removal function with the PCR amplification process itself by incorporating duplex-specific nucleases into the amplification reaction. This allows simultaneous amplification of unique sequences and removal of repetitive sequences in a single integrated process, eliminating the need for separate affinity chromatography steps and significantly reducing processing time.
4Manufacturing precision
If cross-linking agents are used to prevent hybridization of repetitive sequences, then probe specificity is improved, but the process becomes complex and difficult to reproduce
Solution Approach 1:
The patent replaces chemical cross-linking agents with enzymatic duplex-specific nucleases that naturally and specifically recognize and cleave double-stranded repetitive sequences. This enzymatic approach is more specific, controllable, and reproducible than chemical cross-linking, as enzyme activity can be precisely controlled through temperature and buffer conditions without introducing complex chemical variables.
Data Source
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AI summary
A method for detecting the presence of a target sequence comprising: a. adding a probe, said probe having been produced by a method comprising: i. obtaining double strand polynucleotides known to contain complementary target sequences and repetitive sequences; ii. fragmenting said double strand polynucleotides into fragments; iii. denaturing said fragments into single strands; iv. hybridizing said repetitive sequences to form a mixture of double strands and single strands; v. cleaving said double strands; and vi. amplifying said single strands wherein said single strands are complementary to said target sequences; and b. detecting said probe wherein said detection is selected from a group consisting of ISH, FISH, CGH, spectral karyotyping, chromosome painting, Northern blots, Southern blots, microarray analysis, and combinations thereof.