Single Cell Genome Analysis Using Housekeeping Gene Detection
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Solution Overview
Problem
Current methods for analyzing single cell genomes in higher organisms are inefficient due to the lack of effective strategies for whole-genome amplification, which often result in unsuccessful amplification and subsequent sequencing issues.
Innovation Solution
A method involving the separation and lysis of single cells to obtain whole-genome DNA, followed by whole-genome amplification using techniques like MDA or DOP-PCR, and subsequent PCR amplification using housekeeping-gene-specific primers to detect uniform distribution and ensure amplification product quality for sequencing.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Quantity of substance
If whole-genome amplification is performed on single cell DNA, then sufficient DNA amount for sequencing is obtained, but amplification success rate is low due to interfering factors
Solution Approach 1:
The patent performs preliminary quality assessment of the amplification product before sequencing by detecting housekeeping genes and evaluating uniform distribution across chromosomes. This preliminary action identifies successful amplifications early, preventing wasted sequencing resources on failed samples and effectively increasing the overall success rate of the workflow.
Solution Approach 2:
The patent implements a feedback mechanism where the quality of amplification products is assessed through housekeeping gene detection and uniform distribution analysis. This feedback information is used to determine whether to proceed with sequencing, allowing optimization of the amplification process and selection of successful samples for downstream applications.
2Ease of manufacture
If conventional DNA analysis methods are used, then known sites can be studied, but they cannot effectively study new species genomes or provide comprehensive genome coverage
Solution Approach 1:
The patent employs whole-genome amplification followed by high-throughput sequencing, a universal approach that can study any genome regardless of whether it is from a known or new species. The method provides comprehensive genome coverage and can detect various genetic variations, making it highly versatile for different research applications while maintaining ease of operation through standardized protocols.
3Productivity
If single cell genome sequencing is performed without quality assessment, then sequencing can be conducted, but unsuccessful amplification samples waste sequencing resources
Solution Approach 1:
The patent performs preliminary quality assessment of the amplification product before sequencing by detecting housekeeping genes and evaluating uniform distribution across chromosomes. This preliminary action identifies successful amplifications early, preventing wasted sequencing resources on failed samples and effectively increasing the overall success rate of the workflow.
Solution Approach 2:
The patent implements a feedback mechanism where the quality of amplification products is assessed through housekeeping gene detection and uniform distribution analysis. This feedback information is used to determine whether to proceed with sequencing, allowing optimization of the amplification process and selection of successful samples for downstream applications.
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 effectively produces a whole-genome amplification product that meets sequencing requirements, allowing for the construction of a genome sequence library and high-throughput sequencing, thereby obtaining accurate DNA sequence information for deeper analysis of single cell genomes.
Implementation Method 1
the single cell genome needs to be subjected to a whole-genome-amplification (WGA) to achieve a sufficient amount
Implementation Method 2
performing a PCR amplification using the whole-genome amplification product as template and using housekeeping-gene-specific primers to detect the housekeeping gene
Data Source
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AI summary
A method for analyzing a genome of a single cell is provided, and a kit is also provided. The method for analyzing the genome of the single cell may comprise separating and lysing the single cell to obtain a whole-genome DNA of the cell; subjecting the whole-genome DNA to a whole-genome amplification to obtain a whole- genome amplification product; performing a PCR amplification using the whole-genome amplification product as template and using housekeeping-gene-specific primers to detect the housekeeping gene of the whole-genome amplification product; and determining whether the whole genome amplification product meets a requirement for sequencing based on the detection result, wherein a uniform distribution of the amplification product in each chromosome is an indication of the amplification product meeting the requirement for sequencing.