DNA Probe Design via High-Concentration Random Primer PCR
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Solution Overview
Problem
Current methods for producing DNA libraries and probes are not sufficiently convenient or reproducible, limiting their application in genomic analysis, particularly for organisms with large genomes, and often require costly and complex procedures.
Innovation Solution
A method involving PCR with a random primer at a designated concentration to produce a DNA library, allowing for the design of nucleotide sequences for DNA probes with high reproducibility, enabling accurate and cost-effective genomic analysis.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If restriction enzyme treatment and adaptor ligation are used to reduce genome complexity, then DNA marker determination becomes feasible, but the number of processes and cost increase
Solution Approach 1:
The invention extracts only the necessary components for DNA marker determination by using PCR to directly amplify and determine specific regions of genomic DNA, eliminating the need for restriction enzyme treatment and adaptor ligation steps while maintaining the capability to obtain reliable DNA markers
Solution Approach 2:
Instead of reducing genome complexity through restriction enzyme digestion before amplification, the invention inverts the approach by using PCR with carefully designed primers to selectively amplify target regions directly from genomic DNA, achieving the same goal through a reversed procedural sequence
2Reliability
If random primers are used at low concentration for PCR amplification, then genome complexity is maintained, but reproducibility of amplification is poor
Solution Approach 1:
The invention changes the concentration parameter of random primers from low to high (1-10 μM range), which fundamentally alters the PCR amplification dynamics to achieve both high reproducibility and efficient amplification of genomic DNA regions
Solution Approach 2:
The invention introduces dynamic control of amplification by using high concentration random primers that can adaptively bind to various genomic regions, allowing the system to dynamically select and amplify multiple target sequences simultaneously with consistent reproducibility
3Ease of manufacture
If entire genomic region is amplified uniformly, then genomic library can be produced, but complexity of genomic DNA cannot be reduced
Solution Approach 1:
The invention segments the genomic DNA into specific amplifiable regions by using high concentration random primers that target discrete sequences throughout the genome, producing a genomic library composed of manageable fragment segments rather than attempting to handle the entire complex genome at once
Solution Approach 2:
The invention applies partial action by amplifying only specific regions of the genome that are accessible to the random primers, rather than attempting to amplify the entire genome uniformly, thereby producing a usable genomic library without the need to manage the full complexity of genomic DNA
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
The method achieves high reproducibility and cost-effectiveness in producing DNA probes suitable for genetic analysis, such as genetic linkage analysis, by uniformly amplifying genomic DNA across the entire genome.
Implementation Method 1
a random primer at a high concentration is used in a reaction solution, and a nucleic acid amplification reaction is carried out
Implementation Method 2
nucleic acid amplification reaction is carried out in the reaction solution, and a nucleotide sequence of a DNA probe used for detecting an amplified nucleic acid fragment is designed on the basis of the nucleotide sequence of the amplified nucleic acid fragment
Data Source
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AI summary
This invention provides a DNA probe that is applicable to a DNA library prepared in a simple manner with excellent reproducibility. Such DNA probe is produced by a method comprising steps of performing a nucleic acid amplification reaction in a reaction solution containing genomic DNA and a random primer at a high concentration, so as to obtain a DNA fragments with the use of the genomic DNA as a template; determining the nucleotide sequence of the resulting DNA fragments; and, on the basis of the nucleotide sequence of the DNA fragments obtained in the step above, designing a DNA probe used for detecting a DNA fragment.