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High resolution, high throughput hla genotyping by clonal sequencing

A genotype, HLA-A technology, applied to the determination/testing of microorganisms, biochemical equipment and methods, etc., can solve the problems of phase ambiguity retention, time-consuming, etc.

Inactive Publication Date: 2011-07-13
F HOFFMANN LA ROCHE & CO AG
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  • Summary
  • Abstract
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  • Claims
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AI Technical Summary

Problems solved by technology

However, ambiguity in HLA typing data may be preserved due to multiple polymorphisms between alleles and resulting phase ambiguity when both alleles are amplified and sequenced together
Resolving this ambiguity requires time-consuming methods, e.g., separately amplifying and then analyzing both alleles

Method used

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  • High resolution, high throughput hla genotyping by clonal sequencing
  • High resolution, high throughput hla genotyping by clonal sequencing
  • High resolution, high throughput hla genotyping by clonal sequencing

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Embodiment Construction

[0073] multiplex pyrosequencing

[0074] Analysis of multiple HLA loci across multiple samples in a single 454 run is facilitated by the incorporation of molecular ID (MID) tags into the PCR primers. Table 1 shows the sequence of the 454 HLA-specific fusion primer with adapter sequence (for bead capture) and 4-base MID tag. Additional sequences giving 5-base MID tags are provided.

[0075] In preliminary experiments, 24 cell lines with known HLA genotypes (Table 2) were analyzed. In subsequent experiments, 48 ​​samples were analyzed.

[0076] Fifteen primer pairs were designed for exons 2, 3 and 4 of HLA-A, B and C loci, exons 2 of DRB1, DPB1, DPA1, DQA1, and exons 2 and 3 of DQB1 . Primers with twelve different MID tags were designed for each target sequence, a total of 180 (15 x 12). Primers for exon 2 of DRB1 also amplified the DRB3, DRB4 and DRB5 loci, which are genes present on specific DRB1 haplotypes. After amplification of various samples, PCR products were analy...

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Abstract

The invention provides methods and reagent for performing full, multi-locus HLA genotyping for multiple individuals in a single sequencing run using clonal sequencing.

Description

Background of the invention [0001] Class I and class II HLA loci are the most polymorphic genes in the human genome, and the complex pattern of patch polymorphism is mainly localized in exon 2 of class II genes and exons 2 and 3 of class I genes middle. Allele-level resolution of HLA alleles that are clinically important for hematopoietic stem cell transplantation is technically challenging with current HLA typing methods. Several large-scale studies have demonstrated precise, allele-level HLA differentiation between donor and patient by reducing the incidence and severity of acute and chronic graft-versus-host disease and improving the rate of successful engraftment Matching significantly improved overall graft survival. For example, when 8 of the 8 most prominent HLA loci were matched, compared with 6 of 8, post-transplant survival was 60% better after 12 months. [0002] Current practice is to maintain a bone marrow donor registry in which millions of potential donors ar...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
CPCC12Q1/6881C12Q1/6855C12Q2600/156C12Q2600/16C12Q2563/179C12Q2525/149C12Q2565/537
Inventor R·G·希古奇G·本特利H·A·埃利克
Owner F HOFFMANN LA ROCHE & CO AG
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