Single-Cell Fetal DNA Amplification via Two-Stage Primer Design
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Solution Overview
Problem
Current methods for determining a genetic condition of a fetus, such as whole genome amplification, face challenges in maintaining amplification consistency and accuracy, leading to biased PCR products and limited ability to uniformly amplify specific gene regions from a single cell.
Innovation Solution
A method involving the design of a primer set for PCR that employs local and global alignment scores to select primers with reduced complementarity, ensuring efficient amplification of objective gene regions from a single cell, including steps for primer selection, amplification, and sequencing.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Quantity of substance
If whole genome amplification is performed to increase the amount of DNA from a single cell, then the trace amount of genomic DNA is remarkably increased, but the amplification factor cannot be constantly maintained and PCR product becomes biased
Solution Approach 1:
The patent segments the amplification process into two distinct stages: a first amplification stage that generates initial DNA copies, and a second amplification stage that produces the final PCR products. This segmentation allows each stage to be optimized independently, with the first stage focusing on generating sufficient material and the second stage focusing on maintaining amplification consistency and uniformity, thereby resolving the contradiction between increasing DNA quantity and maintaining amplification precision.
2Reliability
If a single cell is isolated and DNA is extracted to determine genetic condition, then the possibility of miscarriage is eliminated, but the amount of DNA obtained is extremely limited requiring whole genome amplification
Solution Approach 1:
The patent performs preliminary action by conducting the first amplification stage before the second amplification stage. This preliminary amplification generates sufficient DNA material from the single cell, ensuring that there is enough template for subsequent specific gene region amplification. This resolves the contradiction by preparing adequate DNA quantity in advance while maintaining the safety benefit of single-cell analysis.
3Productivity
If multiplex PCR is used to amplify multiple target regions simultaneously, then efficiency is improved, but primer design becomes complex and nonspecific products increase
Solution Approach 1:
The patent segments the amplification targets into multiple specific gene regions, each with its own optimized primer pair. By dividing the complex multiplex amplification into manageable specific targets and using the two-stage amplification approach, the patent maintains high specificity for each target while achieving efficient simultaneous amplification of multiple regions, thus resolving the contradiction between productivity and manufacturing precision.
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 allows for accurate and uniform amplification of specific gene regions, enhancing the reliability and efficiency of determining genetic conditions by reducing nonspecific products and improving the analysis of genetic abnormalities from a single cell.
Implementation Method 1
an amplification step performed through a polymerase chain reaction in which the objective region is amplified through the polymerase chain reaction using a primer set designed so as to amplify the objective region through the polymerase chain reaction using the genomic DNA extracted from the single cell as a template
Data Source
AI summary
Provided is a method for determining a genetic condition of a fetus, the method including: an objective region selection step of selecting an objective region for determining the genetic condition, from regions on a human genome; a step of isolating a single cell from a maternal blood sample; a step of extracting genomic DNA from the single cell; a step of performing PCR amplification on the objective region using a primer set designed so as to perform the PCR amplification on the objective region using genomic DNA extracted from the single cell as a template; and a DNA sequencing step of decoding a DNA base sequence of a PCR amplification product of the objective region, in which the primer set designed so as to perform the PCR amplification on the objective region is designed through a method for designing a primer set used for the polymerase chain reaction, the method for designing a primer set including a first stage selection step based on a local alignment score and a second stage selection step based on a global alignment score.