Genome Integrity Assessment via Multi-Primer PCR and Gel Electrophoresis
Solution Overview
Problem
Current methods for assessing the integrity of genomic DNA from single cells, particularly after deterministic restriction site whole genome amplification (DRS-WGA), lack specific assays to evaluate genome integrity and predict the quality of the amplification product, which is crucial for downstream molecular assays.
Innovation Solution
A method and kit using multiple primer pairs to amplify specific DNA sequences from the DRS-WGA product, allowing for the assessment of genome integrity and predicting the success of molecular assays through PCR product detection and agarose gel electrophoresis, with primer pairs targeting different chromosome arms and fragment lengths.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If conventional WGA methods are used to amplify single cell DNA, then amplification can be achieved, but the methods lack specific assays to evaluate genome integrity and predict quality of the amplification product
Solution Approach 1:
The patent divides the genome into specific chromosomal regions (at least three different chromosome arms) and designs primer pairs targeting each region. This segmentation allows independent assessment of different genomic areas, providing comprehensive genome integrity evaluation through multiple discrete PCR reactions rather than a single complex assay.
Solution Approach 2:
The patent performs PCR amplification and agarose gel electrophoresis as preliminary quality control steps before downstream molecular assays. By assessing genome integrity and predicting amplification quality in advance, the method allows researchers to determine whether samples are suitable for subsequent expensive and time-consuming molecular analyses, preventing waste of resources on degraded samples.
2Reliability
If DRS-WGA is used to amplify DNA from fixed cells, then tolerance to DNA degradation is improved, but there are still no specific assays to evaluate the quality of the DRS-WGA product
Solution Approach 1:
The patent implements a feedback mechanism by using the results of PCR amplification and gel electrophoresis to assess the quality of DRS-WGA products. The presence, absence, and intensity of expected PCR products from different chromosome arms provide feedback on whether the DRS-WGA successfully amplified genomic DNA of sufficient quality, allowing researchers to make informed decisions about proceeding with downstream applications.
Solution Approach 2:
The patent replaces complex mechanical or biochemical quality assessment methods with a simpler PCR-based molecular assay. Instead of using physical measurements or complex biochemical tests to evaluate DRS-WGA product quality, the method uses specific primer pairs and standard PCR amplification followed by gel electrophoresis, which are routine and easily interpretable techniques.
3Ease of operation
If only concentration measurement is used to assess WGA product quality, then the assessment is simple, but it cannot predict the success of downstream molecular assays
Solution Approach 1:
The patent merges multiple assessment functions into a unified PCR-based quality control protocol. By combining genome integrity assessment, amplification quality prediction, and suitability evaluation for downstream applications into a single set of PCR reactions and gel electrophoresis steps, the method maintains operational simplicity while significantly improving measurement precision and predictive accuracy compared to concentration measurement alone.
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 provides robust and reliable assessment of genome integrity and quality, enabling prediction of successful molecular assays such as metaphase and array comparative genomic hybridization, Sanger sequencing, and quantitative PCR, even in samples with damaged DNA.
Implementation Method 1
a first primer pair which hybridises to a DNA sequence of the library having a length from 1000 bp to 5000 bp
Implementation Method 2
the library of DNA sequences is amplified by PCR using at least one first primer pair
Implementation Method 3
agarose gel electrophoresis, with primer pairs targeting different chromosome arms and fragment lengths
Data Source
AI summary
A method for determining the integrity of the genome of a sample and/or the quality of a library of DNA sequences obtained by deterministic restriction site whole genome amplification can include (a) amplifying the library of DNA sequences to produce first, second, and third PCR products each of a different size from 50 bp to 1000 bp, by PCR using at least one first primer pair, one second primer pair and one third primer pair, the primer pairs each hybridizing to a DNA sequence of the library having a length from 1000 bp to 5000 bp and corresponding to a sequence of the genome located respectively on a first, second and third chromosome arm; (b) detecting the first, second and third PCR products; (c) correlating the presence of the first, second and third PCR products with the integrity of the genome of the sample and/or the quality of the library.