Simultaneous KIR Gene Typing via Multi-Primer PCR
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Solution Overview
Problem
Current methods for sequence-based typing of Killer Cell Immunoglobulin-like Receptor (KIR) genes are limited in their ability to identify all alleles, especially null alleles, due to extensive sequence homology and the complexity of KIR gene structure, leading to ambiguous allele combinations and lengthy PCR amplification times.
Innovation Solution
A simultaneous sequence-based typing method for all 14 functional KIR genes is developed, using a scientific PCR amplification strategy with multiple pairs of gene-specific primers to amplify coding sequences under uniform thermocycling parameters, allowing for simultaneous PCR amplification and sequencing of all KIR genes.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If conventional PCR amplification methods are used for KIR genes, then amplification can be achieved, but amplification time is lengthy and non-specific amplification occurs
Solution Approach 1:
The patent divides the amplification process into multiple sequential PCR steps with different primer pairs. Each primer pair targets specific exons or regions of KIR genes, allowing systematic amplification of all 14 functional KIR genes while maintaining specificity and reducing total amplification time through optimized cycling conditions for each segment.
2Productivity
If multiple KIR genes are amplified simultaneously, then throughput is improved, but sequence homology causes ambiguous allele combinations
Solution Approach 1:
The patent employs gene-specific primer pairs that target unique regions or exons of each KIR gene. By designing primers with local specificity to distinguish between highly homologous genes (e.g., KIR2DL1-2DL5 family), the method achieves simultaneous amplification of all 14 functional genes while maintaining accurate allele identification through localized sequence differentiation.
3Measurement precision
If KIR gene structure complexity is considered, then accurate typing is achieved, but method complexity increases
Solution Approach 1:
The patent addresses KIR gene structure complexity (9 exons, 8 introns, variable domains) by segmenting the amplification strategy into multiple PCR steps, each targeting specific exon combinations. This systematic segmentation simplifies the overall process by breaking down the complex 14-gene amplification into manageable, optimized reactions while maintaining comprehensive coverage of all functional genes.
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 method enables high-throughput, cost-effective, and efficient identification of all KIR alleles, overcoming the limitations of existing techniques by reducing amplification time and preventing non-specific amplification, thus facilitating population genetics, tissue typing, and disease-associated studies.
Implementation Method 1
using a scientific PCR amplification strategy with multiple pairs of gene-specific primers to amplify coding sequences under uniform thermocycling parameters
Data Source
AI summary
Based on the structural features of KIR full genomic sequences, the distribution of single nucleotide polymorphisms in their coding regions and the length of flanking intronic sequence of each exon, a method for high-throughput simultaneous sequence-based typing of all the 14 functional killer cell immunoglobulin-like receptor (KIR) genes is disclosed including: developing a scientific and reasonable polymerase chain reaction (PCR) amplification strategy; simultaneously amplifying the complete coding sequence of each functional KIR gene using 3˜5 pairs of KIR gene-specific PCR primers that have similar annealing temperature; and determining the nucleotide sequences of the exons carried by each PCR amplicon in both directions using the forward and reverse sequencing primers, respectively, as shown in FIG. 1.