DNA Sequencing as a Tissue-Typing Tool

The ever increasing numbers of reported HLA alleles pose a problem to the modern tissue typing laboratory (Table 1 ) (1 -4 ). PCR-sequencing based typing (PCR-SBT) analyzes the complete nucleotide sequence of the polymorphic exons of HLA class I and II and, as such, provides the highest resolution available for HLA typing which includes the complete characterization of new alleles. Sequencing using Taq DNA polymerase, or “cycle sequencing,” is essentially PCR in which one primer and the enzyme have been modified to ensure even incorporation of each ddNTP into the growing DNA strand. Such even incorporation is important for the detection of heterozygous bases where two different nucleotides are present. The methods described here have been developed and used on a slab gel automated DNA sequencer (Applied Biosystems 373) and they are now being used on a 16-capillary automated DNA sequencer (Applied Biosystems 3100 Genetic Analyzer).

Locus	No. of alleles 1996	No. of alleles 1998	No. of alleles 2002
HLA-A	78	119	246
HLA-B	173	245	481
HLA-C	42	74	118
HLA- DPB1	77	85	99
HLA-DQB1	31	39	53
HLA-DRB1	162	201	325