[Development of a high-throughput sequence-based typing assay for human leukocyte antigen loci].
OBJECTIVE
To develop a reliable assay for simultaneous sequence-based typing (SBT) of HLA-DPA1 and HLA-DPB1, and to apply it for the study of allelic polymorphisms in southern Chinese Han population.


METHODS
Based on full-length HLA-DPA1 and HLA-DPB1 allelic sequences, locus-specific PCR primers were designed and applied to amplify the target sequence encompassing the entire exon 2 of HLA-DPA1 and HLA-DPB1. PCR products were purified with magnetic beads, and run through an ABI 3730 DNA sequencer. Genotypes were assigned with an Assign 3.5 SBT software.


RESULTS
The target sequences of HLA-DPA1 and HLA-DPB1 were both amplified with the PCR procedure. Little background and noise was observed in the derived sequences. Among 176 non-related healthy individuals, 4 HLA-DPA1 alleles with the frequencies of DPA1*02:02 (0.589) > DPA1*01:03 (0.284) > DPA1*02:01 (0.096) > DPA1*04:01 (0.031) were identified. In addition, 14 HLA-DPB1 alleles, including 4 common alleles (with a frequency of more than 5%, namely DPB1*05:01, DPB1*02:01, DPB1*04:01 and DPB1*02:02), 7 alleles with a frequency ranging from 1%-5% and 3 alleles with a frequency of less than 1% were identified. The results of HLA-DPB1 genotyping were all in accordance with the typing results derived from an Atria AlleleSEQR HLA-DPB1 kit.


CONCLUSIONS
A reliable technique has been established for simultaneous genotyping of HLA-DPA1 and HLA-DPB1, which may have a broad application in population and disease association studies.
Shenzhen Blood Center, Shenzhen, Guangdong 518035, P. R. China. zhihui_deng@yahoo.com.cn
Qiong
Q
Yu
Da-ming
DM
Wang
Zhi-hui
ZH
Deng
D000483:Alleles; D001483:Base Sequence; D016172:DNA Fingerprinting / Q000379:methods*; D005787:Gene Frequency; D005838:Genotype; D059867:HLA-DP alpha-Chains / Q000235:genetics*; D059868:HLA-DP beta-Chains / Q000235:genetics*; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D006801:Humans; D008969:Molecular Sequence Data; D011110:Polymorphism, Genetic
D059867:HLA-DP alpha-Chains; D059868:HLA-DP beta-Chains; C071172:HLA-DPA1 antigen; C071170:HLA-DPB1 antigen