HLA Typing via Simultaneous PCR and Phase Resolution
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Solution Overview
Problem
Conventional DNA typing methods for HLA genes face challenges in accurately determining polymorphic regions and cis/trans positional relationships, leading to phase ambiguity and failure in detecting null alleles, especially when PCR conditions for each gene locus are not unified.
Innovation Solution
Designing sets of primers that specifically hybridize to upstream and downstream regions of HLA genes, allowing for simultaneous PCR amplification under the same conditions, and selecting a suitable DNA polymerase to eliminate phase ambiguity and enable high-speed PCR.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If conventional DNA typing methods are used for HLA genes, then the typing process can be performed, but phase ambiguity occurs and null alleles cannot be detected due to inability to determine cis/trans positional relationships
Solution Approach 1:
The HLA gene region is divided into multiple specific loci (HLA-A, HLA-B, HLA-C, HLA-DRB1, HLA-DQB1, etc.), and separate primers are designed for each locus to amplify them individually. This segmentation allows tracking of each allele's origin and determines cis/trans relationships, eliminating phase ambiguity.
Solution Approach 2:
A standardized primer design system acts as an intermediary between the complex HLA gene region and the sequencing analysis. The primers include standardized regions that facilitate consistent amplification and sequencing across multiple loci, enabling accurate phase determination and null allele detection.
2Productivity
If PCR conditions are optimized for each individual HLA gene locus, then amplification efficiency is improved, but the operation becomes complex and time-consuming
Solution Approach 1:
The primer design provides universality by including standardized regions that work across multiple HLA loci with the same PCR conditions. The primers are designed to bind to conserved regions while maintaining locus-specific amplification, allowing a single set of PCR conditions to amplify multiple different HLA genes simultaneously.
Solution Approach 2:
The primers are designed with specific parameter optimizations including standardized annealing temperatures and extension times that work across all target loci. By carefully selecting primer sequences with similar melting temperatures and designing them to amplify products of comparable lengths, the same PCR cycling parameters can be used for all HLA loci.
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 precise and rapid typing of HLA genes, including detection of null alleles, by determining all nucleotide sequences required for DNA typing from a single molecule, thereby improving matching accuracy in transplantation and simplifying the PCR operation.
Implementation Method 1
preparing sets of primers which respectively hybridize specifically to an upstream region and a downstream region of at least two genes selected from genes belonging to HLA class I and class II
Implementation Method 2
amplifying at least two genes from a test sample (DNA) at the same time under the same PCR conditions in a single container
Data Source
AI summary
The purpose of the present invention is to provide a method and kit for highly precise DNA typing, in which a high throughput sequencer is used and ambiguity derived from phase ambiguity is eliminated. The present invention provides a method for the DNA typing of HLA, which is characterized by comprising: (1) a step of preparing sets of primers which respectively hybridize specifically to an upstream region and a downstream region of at least 2 genes selected from genes belonging to HLA class I and HLA class II in a human genome sequence, and are capable of amplifying under the same PCR conditions; (2) a step of simultaneously amplifying said at least 2 genes in a test sample (DNA) using the sets of primers in a single container under the same PCR conditions; (3) a step of determining the nucleotide sequences of PCR amplified products; and (4) a step of optionally carrying out a homology search within a database.