High-resolution HLA-DPB typing based upon computerized analysis of data obtained by fluorescent sequencing of the amplified polymorphic exon 2.
To differentiate 32 HLA-DPB alleles, conventional techniques such as serology and cellular typing are inadequate for high-resolution DPB typing. The most refined DNA typing until now is SSO typing and new selected oligonucleotides can be added to this system to distinguish new allele sequences. DNA sequencing, however, reveals directly the sequence information of all polymorphic HVRs and has the advantage of being independent from exon polymorphisms. We have developed a new DNA-based typing approach that is rapid, fully automated, and therefore suitable for routine typing. The system is based upon direct sequencing of amplified DNA with fluorescent-labeled primers. The designation of alleles is obtained by a comparison of all polymorphic positions in the determined sequence with all known allele sequences retained in a database along with their heterozygous combinations. Sequence data at both constant and polymorphic positions are used for quality control. In this study, the typing results of a panel of 91 previous SSO-typed DNA samples are described. After comparison with the SSO-typing results, we conclude that with this SBT system allele assignment is reliable. The method is easy to perform since both sequencing and assignment are automated. Furthermore, the system is easily applicable to other gene systems.
Versluis
L F
LF
Diagnostic DNA Laboratory, Utrecht Academic Hospital, The Netherlands.
Rozemuller
E
E
Tonks
S
S
Marsh
S G
SG
Bouwens
A G
AG
Bodmer
J G
JG
Tilanus
M G
MG
D001483:Base Sequence; D005091:Exons; D005456:Fluorescent Dyes; D005838:Genotype; D006682:HLA-DP Antigens / Q000145:classification* / Q000235:genetics; D059868:HLA-DP beta-Chains; D006801:Humans; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods; D011110:Polymorphism, Genetic
D005456:Fluorescent Dyes; D006682:HLA-DP Antigens; D059868:HLA-DP beta-Chains; C071170:HLA-DPB1 antigen