In Situ Hybridization Probes via Selective PCR Amplification
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Solution Overview
Problem
Current methods for producing ISH probes for chromosomal aberration diagnosis face challenges due to non-specific hybridizations caused by repetitive sequences, leading to high background noise and incomplete removal of repetitive elements, which complicates the evaluation of chromosomal aberrations in tumor cells.
Innovation Solution
A method involving the design and synthesis of scythe and antisense primer pairs for PCR, followed by multiplex PCR to amplify specific nucleic acid sequences without repetitive parts, allowing for the production of probes with reduced or no repetitive sequences, enabling clearer in situ hybridization signals.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Quantity of substance
If classic methods (BAC, YAC, cosmids, fosmids) are used to produce ISH probes containing large areas of genomic DNA, then the probe coverage is sufficient for chromosomal aberration detection, but repetitive sequences and pseudogenes cause non-specific hybridizations and high background noise
Solution Approach 1:
The patent extracts and removes repetitive sequences, pseudogenes, and paralogous sequences from genomic DNA to create probes that contain only unique sequences. This is achieved through specialized PCR conditions and purification steps that selectively eliminate repetitive elements while retaining the necessary probe coverage for chromosomal aberration detection.
Solution Approach 2:
The patent segments the genomic DNA into smaller, controlled PCR fragments with specific size ranges. By fragmenting the DNA and selecting fragments within optimal size ranges, the method achieves sufficient coverage while avoiding large repetitive regions that would cause background noise in hybridization.
2Object-affected harmful factors
If blocking DNA (Cot-1 DNA, Salmon Sperm DNA) is added in high excess to avoid background from repetitive sequences, then non-specific hybridizations are reduced, but signals from repetitive sequences cannot be completely avoided and evaluation remains difficult
Solution Approach 1:
The patent performs preliminary removal of repetitive sequences during probe production through controlled PCR amplification and purification steps. By eliminating repetitive sequences before hybridization rather than attempting to block them during hybridization, the method achieves both reduced non-specific binding and improved signal evaluation without requiring blocking DNA.
3Object-affected harmful factors
If BAC clones are fragmented and processed through multiple PCR and digestion steps to remove repetitive sequences, then the probe is freed from repetitive sequences, but the procedures involve considerable effort and are never complete or safe
Solution Approach 1:
The patent optimizes PCR parameters including temperature profiles, buffer compositions, and cycle conditions to selectively amplify unique sequences while suppressing repetitive sequence amplification. By carefully controlling PCR parameters, the method achieves complete removal of repetitive sequences in a single or few amplification steps rather than multiple iterative processes.
Solution Approach 2:
The patent uses PCR to create multiple copies of the desired unique sequence fragments directly from the genomic DNA. This amplification approach replaces the need for cloning into BAC vectors and subsequent manual processing, achieving both repetitive sequence removal and probe production in a streamlined manner.
4Object-affected harmful factors
If PCR is used to produce probes from specific areas free of repetitive sequences, then non-repetitive probes are obtained, but the coverage is often not great and several megabases are required for clinically relevant ISH probes
Solution Approach 1:
The patent combines multiple PCR-amplified unique sequence fragments into a single probe mixture that collectively covers several megabases of genomic DNA. By pooling numerous small unique fragments, the method achieves both the repetitive sequence freedom of PCR-based approaches and the extensive coverage required for clinically relevant chromosomal aberration detection.
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 results in probes that provide clearer and more specific in situ hybridization signals, reducing background noise and enhancing the diagnostic accuracy of chromosomal aberrations, particularly in tumor cells.
Implementation Method 1
design and synthesizing of scythe and Antisense-Primer pairs for a polymerase chain reaction on selected specific nucleic acid sequences
Implementation Method 2
primers which hybridize to the non-complementary linker sequences
Data Source
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AI summary
The invention relates to a kit, to probe mixtures, and to probes for detecting a chromosomal abnormality. A gene probe, obtained by means of a method having the following steps: (a) examining genomic sections for sequence ranges having non-repetitive nucleic acid sequences and selecting one or more nucleic acid sequences; (b) designing and synthesizing primer pairs for a polymerase chain reaction on the non-repetitive nucleic acid sequences, wherein the primers each have an oligonucleotide sequence complementary to the strand or antisense strand of the non-repetitive nucleic acid sequence, and a non-complementary universal linker sequence; (c) performing a first PCR and obtaining a first mixture (Pool A) of nucleic acid fragments; (d) performing a multiplex PCR on the mixture (Pool A) by using primers that hybridize to the linkers and obtaining a mixture (Pool B) having reproduced non-repetitive nucleic acid fragments that are suitable for chromogenic or fluorescent in situ hybridization of chromosomes (FISH/CISH/ISH).