HLA-DQB1 sequencing-based typing using newly identified conserved nucleotide sequences in introns 1 and 2.
Sequencing-based typing (SBT) human leukocyte antigen (HLA) class I and II genes should examine entire exon sequences where polymorphisms lie. Primers for the amplification of complete exons therefore anneal in introns and their design relies on accurate intron sequences being available. We decided to develop a SBT method for HLA-DQB1 using amplification primers which anneal in introns 1 and 2, yet the amount of intron sequence data previously available in databases was sparse. Therefore, we undertook a systematic sequencing of introns 1 and 2 using DNA from cell lines homozygous for DQB1. This study confirmed an earlier report that the non-coding regions of this gene are the most polymorphic seen in the human genome. Intron sequences within an allele group were largely identical, the exceptions being DQB1*0301 differing from other DQB1*03 allele groups and DQB1*0601 differing from all other DQB1*06 alleles. A retroviral Alu element, related to the AluYa5a2 subfamily, was identified uniquely inserted in intron 2 of DQB1*02 alleles. For the typing approach, six amplification primers were designed based on conserved allele group sequences covering all of the HLA DQB antigens, and two sequencing primers were also designed which anneal in intron 2. This method has proved to be very robust and has been used as part of a referral DNA sequencing service for a number of years.
Dunn
P P J
PP
DNA Reference Laboratory, National Blood Service, Southmead Road, Bristol BS10 5ND, UK. paul.dunn@nzblood. co.nz
Day
S
S
Williams
S
S
Bendukidze
N
N
D000483:Alleles; D001483:Base Sequence; D017124:Conserved Sequence; D014644:Genetic Variation; D006683:HLA-DQ Antigens / Q000145:classification / Q000235:genetics*; D059866:HLA-DQ beta-Chains; D006650:Histocompatibility Testing / Q000379:methods*; D006801:Humans; D007438:Introns; D008969:Molecular Sequence Data; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D011110:Polymorphism, Genetic; D017422:Sequence Analysis, DNA
D006683:HLA-DQ Antigens; D059866:HLA-DQ beta-Chains; C069051:HLA-DQB1 antigen