DNA Profiling Assay Using Deletion-Insertion Polymorphisms
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Solution Overview
Problem
Current DNA profiling methods, particularly in forensic and genetic analysis, face challenges with high variability in genetic loci, leading to ambiguous amplification products and difficulties in separating multiple loci, especially with degraded DNA samples and the need for precise identification of single nucleotide polymorphisms.
Innovation Solution
A DNA profiling assay using simultaneous polymerase chain reaction amplification of at least 30 loci characterized by deletion-insertion polymorphisms (DIPs), where each locus has alleles differing by 1-100 nucleotides, utilizing specific primers and fluorescent labels for multiplex detection, enabling accurate differentiation and identification.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If multiple distinct polymorphic genetic loci are amplified simultaneously using STR-based methods, then the throughput and efficiency of DNA profiling is improved, but the amplification products become ambiguous and difficult to separate due to high variability in genetic loci
Solution Approach 1:
The patent changes the type of genetic marker from STR (short tandem repeat) to DIP (deletion-insertion polymorphism). This parameter change in the genetic marker type resolves the contradiction by providing loci with fixed amplicon sizes that can be clearly separated and identified, eliminating the ambiguity problem while maintaining high throughput multiplex amplification of 30+ loci
2Reliability
If the number of loci amplified in a multiplex system is increased to enhance discrimination power, then the reliability of DNA profiling is improved, but the complexity of separating and detecting multiple loci increases
Solution Approach 1:
The patent segments the detection system into multiple fluorescent channels (at least three different fluorescent labels), allowing simultaneous detection of multiple DIP loci with fixed sizes. This segmentation approach enables high discrimination power through analysis of 30+ loci while managing detection complexity through parallel fluorescent channel analysis rather than sequential separation
3Ease of manufacture
If traditional gel electrophoresis systems are used to separate amplification products, then the setup is simple, but the ability to resolve and accurately identify alleles with size differences less than 100 base pairs is insufficient
Solution Approach 1:
The patent replaces the mechanical gel electrophoresis separation system with a DNA sequencing device that uses fluorescent labels and optical detection. This substitution provides superior resolution for identifying alleles with size differences of 1-100 base pairs, accurately determining the precise number of deleted or inserted nucleotides while maintaining operational simplicity through automated sequencing analysis
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 significantly enhances the power of discrimination and throughput in DNA profiling, reducing the risk of laboratory errors and improving the analysis of degraded samples, with a combined probability of identity (CPI) of 2.1 x 10^-13, surpassing existing STR-based methods.
Implementation Method 1
simultaneous amplification of multiple distinct polymorphic genetic loci using the polymerase chain reaction
Implementation Method 2
label one, label two and label three are each different fluorescent labels which can be differentiated and simultaneously detected by a multi-colour detector
Data Source
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AI summary
The present invention relates to a DNA profiling assay comprising the following steps, providing a sample to be analyzed, providing reagents, enzyme and primeroligonucleotides which are necessary for simultaneous polymerase chain reaction amplification of at least 20 loci, amplifying the loci, detecting the amplification products, wherein the amplification products and the loci to be amplified are characterized by the following features, each locus to be amplified is characterized by at least one deletion-insertion polymorphism known to be present in the population, wherein the two alleles from each locus differ in size by more than 2 nucleotides and less than 100 nucleotides, a first set of at least two amplification products ranging in size from about 20 nucleotides to about 300 nucleotides stemming from at least two different loci carries a first label, a second set of at least two amplification products ranging in size from about 20 nucleotides to about 300 nucleotides stemming from at least two different loci carries a second label, a third set of at least two amplification products ranging in size from about 20 nucleotides to about 300 nucleotides stemming from at least two different loci carries a third label, label one, label two and label three are each different fluorescent labels which can be differentiated and simultaneously detected by a multi-colour detector in combination with, e.g. a DNA sequencing device.