Nested PCR Amplification for Non-Invasive Prenatal Paternity Testing
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Solution Overview
Problem
Current non-invasive prenatal paternity testing methods are invasive and carry risks of miscarriage, and existing DNA amplification techniques face challenges such as contamination, allele dropout, and errors in fetal DNA analysis due to low concentrations of fetal cells or DNA in maternal blood.
Innovation Solution
A method involving obtaining genetic material from the alleged father and mother, making genotypic measurements at polymorphic loci, and determining the probability of paternity using a mixed sample of DNA from maternal blood, which includes fetal and maternal DNA, employing techniques like PCR amplification, SNP microarrays, and probabilistic estimation to enrich and analyze fetal DNA.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If chorionic villus sampling or amniocentesis is performed to obtain fetal genetic material, then prenatal genetic diagnosis can be conducted, but the risk of miscarriage increases
Solution Approach 1:
The invention extracts fetal genetic material (cfDNA) from maternal blood plasma without invasive procedures. Fetal cfDNA naturally circulates in maternal blood, and by isolating and analyzing this free-floating DNA, the method achieves prenatal genetic diagnosis while avoiding the miscarriage risks associated with invasive sampling procedures.
Solution Approach 2:
The invention uses maternal blood as an intermediary medium to access fetal genetic material. Instead of directly sampling fetal tissue through invasive procedures, the method analyzes fetal cfDNA that has entered maternal circulation, using maternal blood as a safe intermediary to obtain fetal genetic information.
2Reliability
If fetal cells are isolated from maternal blood to perform genetic analysis, then prenatal diagnosis can be conducted, but the fetal cell concentration is too low (1-40 cells per milliliter) to enable reliable analysis
Solution Approach 1:
The invention replaces mechanical cell isolation methods with molecular amplification techniques. Instead of physically isolating and concentrating fetal cells from maternal blood, the method uses PCR amplification to exponentially increase the amount of fetal cfDNA, converting a physical concentration problem into a chemical amplification process.
Solution Approach 2:
The invention changes the concentration parameter through molecular amplification. By using PCR to amplify fetal cfDNA, the method transforms the extremely low initial concentration of fetal DNA into a sufficient quantity for reliable genetic analysis, effectively changing the concentration parameter without physical concentration steps.
3Quantity of substance
If PCR amplification is used to amplify fetal DNA from mixed maternal and fetal DNA, then sufficient DNA for analysis can be obtained, but contamination and allele dropout errors occur
Solution Approach 1:
The invention segments the amplification process into multiple targeted steps. By designing specific primers for individual SNPs and using a stepwise amplification approach with intermediate purification steps, the method reduces contamination risks and improves the reliability of genotypic measurements compared to single-step whole-genome amplification.
Solution Approach 2:
The invention incorporates quality control feedback mechanisms. By analyzing amplification efficiency and genotypic data quality at intermediate steps, the method can identify and correct potential errors from contamination or allele dropout, improving overall measurement reliability through iterative quality assessment.
4Measurement precision
If multiple SNPs are analyzed simultaneously to determine paternity, then accuracy improves, but the complexity of the amplification and analysis process increases
Solution Approach 1:
The invention uses universal amplification primers that can amplify multiple different SNP targets. By designing primers with universal binding regions combined with SNP-specific regions, the method enables simultaneous analysis of multiple SNPs using a unified amplification platform, reducing overall system complexity while maintaining high measurement 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
Enables non-invasive determination of paternity with high accuracy by overcoming the challenges of low fetal DNA concentrations and contamination, reducing the risk of miscarriage and improving the reliability of fetal DNA analysis.
Implementation Method 1
employing techniques like PCR amplification
Implementation Method 2
amplification of cell-free DNA using ligated adaptors
Data Source
AI summary
Methods for non-invasive prenatal paternity testing are disclosed herein. The method uses genetic measurements made on plasma taken from a pregnant mother, along with genetic measurements of the alleged father, and genetic measurements of the mother, to determine whether or not the alleged father is the biological father of the fetus. This is accomplished by way of an informatics based method that can compare the genetic fingerprint of the fetal DNA found in maternal plasma to the genetic fingerprint of the alleged father.