A generic sequencing based typing approach for the identification of HLA-A diversity.
Sequencing Based Typing (SBT) is a generic approach for the identification of HLA-A polymorphism. This approach includes the high resolution typing of the HLA-A broad reacting groups, HLA-A subtypes and will identify new alleles directly. The SBT approach described here uses a locus specific amplification of DNA from exon 1 to exon 5. The resulting 2,022 bp PCR product serves as a template for the subsequent sequencing reactions. Amplification is followed by direct sequencing of exons 2, 3 and 4 in both orientations with fluorescently labeled primers to define all polymorphic positions leading to a high resolution typing result. In this study the sequence of exons 2 and 3 of a panel of 49 cell lines was determined. In addition, the exon 4 region of 35 cell lines was also sequenced to evaluate the exon 4 polymorphism. The HLA-A type of most of the cells could be identified by sequencing only exons 2 and 3. However, the sequence of exon 4 was required to discriminate A*0201 from A*0209 and A*0207 from A*0215N. In this panel, an identical new "HLA-A*0103" was identified in two Caucasian samples.
Scheltinga
S A
SA
University Hospital Utrecht, Department of Pathology, The Netherlands.
Johnston-Dow
L A
LA
White
C B
CB
van der Zwan
A W
AW
Bakema
J E
JE
Rozemuller
E H
EH
van den Tweel
J G
JG
Kronick
M N
MN
Tilanus
M G
MG
D000483:Alleles; D001483:Base Sequence; D003001:Cloning, Molecular; D005091:Exons; D006580:Genetic Carrier Screening; D015234:HLA-A Antigens / Q000235:genetics* / Q000276:immunology; D006650:Histocompatibility Testing / Q000379:methods*; D006801:Humans; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods*; D011110:Polymorphism, Genetic; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA
D015234:HLA-A Antigens; D015345:Oligonucleotide Probes