Single reaction multiplex PCR primer design for amplifying multiple HLA class I and II genes
AssigneeOne Lambda
InventorsJar-How Lee, Katsuyuki Saito
Life of this patent
Estimating the term…
Abstract
A method providing next generation sequencing (NGS)-based high-resolution HLA typing as a routine clinical test. The method uses a multiplex PCR primer design for amplifying multiple human leukocyte antigen (HLA) Class I and Class II genes in a single reaction for NGS. The test quality is improved and the protocol for typing multiple HLA genes is simplified because the number of amplification reactions are reduced nearly 10-fold, to yield a substantially equimolar ratio of individual HLA gene amplification products. The invention eliminates an amplicon pooling step, reducing the reagent cost, and required sample DNA quantity.
Claims11
A method for genotyping human leukocyte antigen (HLA), the method comprising
- (a) amplifying HLA class I and HLA class II genes in a test sample in a single container to yield at least two amplified products using forward primers and reverse primers,
- wherein the forward primers and the reverse primers specifically hybridize regions which flank at least one gene selected from genes encoding HLA class I antigens HLA-A, HLA-B, HLA-C, and at least one gene selected from genes encoding HLA class II antigens HLA-DQA1 and HLA-DPA1, and a region of exon 2 to a 3′ untranslated region in at least one of HLA-DRB11, HLA-DRB3, HLA-DRB4, HLA-DRB5, HLA-DQB1, and HLA-DPB1, and
- wherein amplifying comprises amplifying by polymerase chain reaction (PCR) comprising a first set of at least 22 amplification cycles, each amplification cycle within the first set comprising a temperature of 98° C. for 10 seconds and 69° C. for 3 minutes, followed by a second set of at least eight amplification cycles, each amplification cycle of the second set comprising a temperature of 98° C. for 10 seconds and 60° C. for 3 minutes;
- (b) determining the nucleotide sequence of the at least one amplified product;
- and
- (c) optionally carrying out a homology search within a database.
A method for DNA typing of human leukocyte antigens (HLA), comprising the steps of
- (a) amplifying with a set of forward primers and reverse primers that specifically hybridize regions which flank at least one gene selected from genes encoding HLA class I antigens HLA-A, HLA-B, HLA-C, and at least one gene selected from genes encoding HLA class II antigens HLA-DQA1 and HLA-DPA1, and a region of exon 2 to a 3′ untranslated region in at least one of HLA-DRB1, HLA-DRB3, HLA-DRB4, HLA-DRB5, HLA-DQB1, and HLA-DPB1,
- wherein amplifying comprises simultaneously amplifying by polymerase chain reaction (PCR) under identical reaction conditions in a single container the at least two genes in a test sample of the at least one of a transplant donor or a transplant recipient using the forward primers and reverse primers;
- and
- wherein the PCR comprises a first set of at least 22 amplification cycles, each amplification cycle within the first set comprising a temperature of 98° C. for 10 seconds and 69° C. for 3 minutes, followed by a second set of at least eight amplification cycles, each amplification cycle of the second set comprising a temperature of 98° C. for 10 seconds and 60° C. for 3 minutes;
- and
- (b) determining the nucleotide sequences of the amplified products.
Dependencies are inferred from the claim text.