Multiplex PCR Kit for Rapidly Mutating Y-STR Discrimination
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Solution Overview
Problem
Current methods for analyzing Y-STRs on the Y chromosome struggle to distinguish between closely related males due to low mutation rates, and existing technologies have not successfully co-amplified multiple rapidly mutating Y-STRs in a single multiplex PCR reaction.
Innovation Solution
A method and kit using a set of primers (SEQ ID NO:1 to 26) that enable the co-amplification of thirteen rapidly mutating Y-STRs at specific loci in a single multiplex PCR reaction, allowing for effective discrimination between individual males even with small DNA samples.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If conventional Y-STR analysis methods are used, then the analysis can be performed with standard protocols, but the methods cannot distinguish between closely related males due to low mutation rates
Solution Approach 1:
The invention changes the parameter of which Y-STR loci are analyzed by specifically selecting rapidly mutating Y-STRs (RM Y-STRs) with mutation rates around 10^-2, compared to conventional Y-STRs with mutation rates around 10^-3. This parameter change in mutation rate enables discrimination between closely related males while maintaining reliable amplification and analysis
2Measurement precision
If multiple Y-STR loci are analyzed separately, then each locus can be analyzed with optimized conditions, but the process requires multiple PCR reactions and increases time and complexity
Solution Approach 1:
The invention merges the analysis of thirteen different RM Y-STR loci into a single multiplex PCR reaction by designing a set of primers that can co-amplify all thirteen loci simultaneously. This combining approach maintains accurate allele determination for each locus while dramatically reducing the number of separate reactions needed
Solution Approach 2:
The primer set designed in the invention has universal applicability across all thirteen RM Y-STR loci, with each primer pair capable of amplifying its specific locus while functioning within the same reaction conditions as the other twelve primer pairs. This multi-functionality allows simultaneous analysis of all loci in one reaction
3Productivity
If multiple Y-STR loci are co-amplified in a single multiplex PCR, then the process time is reduced, but amplification efficiency and precision are compromised
Solution Approach 1:
The invention applies local quality by designing each primer pair with specific characteristics optimized for its target locus, including locus-specific binding sites and fluorophore assignments. Each primer pair maintains its local optimization for specific locus amplification while functioning within the global context of the multiplex reaction
Solution Approach 2:
The invention changes multiple parameters to achieve successful co-amplification: selecting RM Y-STR loci with similar amplification characteristics, optimizing primer concentrations for each locus, choosing fluorophores with non-overlapping emission spectra, and adjusting PCR cycling conditions. These parameter changes enable high-throughput amplification while maintaining precision
4Measurement precision
If male DNA is analyzed in samples containing female DNA, then male DNA can be detected, but contamination risks increase and specific amplification becomes difficult when female DNA significantly outnumbers male DNA
Solution Approach 1:
The invention extracts and amplifies only male-specific DNA sequences by using primers that bind exclusively to Y chromosome loci. This extraction approach isolates the male DNA signal from the background of female DNA, enabling specific detection and reducing the impact of contamination or female DNA overwhelming the reaction
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 solution allows for accurate discrimination of individual males with as little as 65 picograms of male DNA, reducing contamination risks and enabling specific amplification of male DNA in samples with significantly more female DNA, thus improving forensic analysis.
Implementation Method 1
performing polymerase chain reaction (PCR) to co-amplify the RM Y-STRs at loci DYF387S1; DYF399S1; DYF403S1a/b; DYF404S1; DYS449; DYS518; DYS526a/b; DYS547; DYS570; DYS576; DYS612; DYS626; and DYS627 if present in the DNA
Data Source
AI summary
Provided are methods and kits that allow the amplification, in a single multiplex reaction, of the thirteen rapidly mutating Y chromosome short tandem repeats (RM Y-STRs) at loci DYF387S1; DYF399S1; DYF403S1a/b; DYF404S1; DYS449; DYS518; DYS526a/b; DYS547; DYS570; DYS576; DYS612; DYS626; and DYS627, if present in a sample of DNA, and determination of the alleles at these RM Y-STRs. The ability to achieve such determination through a single multiplex arises as a result of a beneficially designed set of primers disclosed herein. Optimised conditions for the PCR also contribute to the advantages observed. Such kits and methods may be of benefit in the context of forensic sciences.