HLA Class II Gene Typing via Segmented PCR and Massively Parallel Sequencing
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Solution Overview
Problem
Current DNA typing methods for HLA genes, such as SBT and Luminex, face challenges in accurately determining polymorphic regions in cis- or trans-configuration, leading to phase ambiguity, especially with fragmented DNA samples in poor preservation states, particularly for HLA class II genes with long introns.
Innovation Solution
A method using a high-throughput massive parallel sequencer to amplify and type HLA class II genes from exon 2 to exon 4, employing specifically designed PCR primers that anneal to intron 1 regions containing exon 2 and exon 3, allowing simultaneous PCR amplification under the same conditions, and subsequent nucleotide sequencing to resolve phase ambiguity.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If conventional DNA typing methods (SBT, Luminex) are used to determine HLA genes, then the typing process can be completed, but phase ambiguity occurs in determining polymorphic regions in cis- or trans-configuration
Solution Approach 1:
The patent segments the HLA gene amplification into two distinct regions: intron 1 region (exon 2 and exon 3) and exon 4 region. By designing separate primer sets for each region and performing sequential amplification, the method captures complete haplotype information including phase relationships, thereby eliminating phase ambiguity while maintaining typing accuracy.
2Reliability
If long amplification regions including intron 1 are targeted for HLA class II genes, then complete haplotype information can be obtained, but amplification efficiency decreases in fragmented DNA samples
Solution Approach 1:
The patent divides the long amplification region into two manageable segments: intron 1 region (exon 2 and exon 3) and exon 4 region. This segmentation reduces the length of each amplification product, improving amplification efficiency in fragmented DNA samples while still capturing complete haplotype information when the segments are combined through sequential amplification.
Solution Approach 2:
The patent performs preliminary amplification of the intron 1 region first, then uses the resulting product as template for subsequent amplification of the exon 4 region. This stepwise preliminary action ensures that even in fragmented samples, the first region is successfully amplified and serves as a foundation for the second amplification step, maintaining overall reliability.
3Loss of time
If multiple HLA genes are amplified simultaneously under the same PCR conditions, then the time required for PCR is shortened, but amplification conditions must be optimized for each gene
Solution Approach 1:
The patent designs universal primer sets that can simultaneously amplify multiple HLA class II genes (HLA-DRB1, HLA-DRB3, HLA-DRB4, HLA-DRB5, HLA-DQB1, HLA-DPB1) under the same PCR conditions. The primers are engineered to target conserved regions across different HLA genes, enabling multi-gene amplification in a single reaction without requiring gene-specific optimization, thus reducing overall PCR time and simplifying the process.
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 eliminates phase ambiguity, enables accurate DNA typing of HLA genes even from poorly preserved samples, reduces amplification region length, and allows for simultaneous PCR amplification of multiple HLA genes, shortening the time required for PCR and data analysis.
Implementation Method 1
a step of preparing a set of primers which anneal specifically to an intron 1 region containing exon 2 and exon 3 and an exon 4 region, respectively
Implementation Method 2
a step of determining the nucleotide sequence of the PCR amplified product using a high throughput massive parallel sequencer
Data Source
AI summary
The present invention addresses the problem of providing a method and kit for the DNA profiling of HLA genes using a high-throughput massively parallel sequencer. The present invention pertains to a method for the DNA profiling of HLA genes, said method being characterized by including: (1) a step for preparing a primer set that anneals specifically to exon 4 and intron 1 and includes exon 2 and exon 3 of at least one target gene selected from the group consisting of HLA-DRB1, HLA-DRB3, HLA-DRB4, HLA-DRB5, HLA-DQB1 and HLA-DPB1 in the base sequence of the human genome; (2) a step for amplifying a sample (DNA) by PCR using the primer set; (3) a step for determining the base sequence of the amplified PCR product; and (4) a step for carrying out a homology search against a database.