Selection of unrelated bone marrow donors by PCR‐SSP typing and subsequent nonradioactive sequence‐based typing for HLA DRB1/3/4/5, DQB1, and DPB1 alleles

Abstract

HLA incompatibility between bone marrow recipients and unrelated donors is one of the main obstacles in bone marrow transplantation. HLA class I and generic class II DR and DQ typing is generally performed by serology. Precise subtyping of HLA class II genes, however, can only be achieved by molecular genetic methods. Here, the final selection of serologically pretyped unrelated bone marrow donors by confirmatory PCR-SSP (PCR-sequence-specific primers) typing and subsequent nucleic acid sequence analysis of the second exon of DRB1, DRB3, DRB4, DRB5, DQB1, and DPB1 alleles is presented. Serologically identical potential marrow donors and their corresponding recipients were analyzed for HLA-DRB identity by PCR-SSP analysis. After solid-phase single-strand separation, direct sequencing of the allele- or group-specific DRB amplified products was performed by applying fluorophorlabelled sequencing primers. Electrophoretically separated sequencing products were detected by means of an automated DNA sequencer. Group-specific amplification and sequencing of DQB1 alleles was carried out for all potential bone marrow donors and recipients, while only the final donor-recipient pair was analyzed for DPB1 alleles. Thus, the presented amplification strategy in combination with direct sequencing of PCR products allows matching of bone marrow transplant pairs with the highest degree of reliability for the assessment of HLA class II identity.

A commented dictionary of techniques for genotyping

“…Stringent reaction conditions and Taq without proof reading activity ensure that only primers fully complementary to the template allow for amplification of the target [129][130][131]. The specific step is primer hybridization and extension in contrast to sole probe hybridization in PCR-SSO [132][133][134]. In primer design, matching the 3'-end of the primer with its template is the most important step.…”

Rapid method for successful HLA class I and II typing from cadaveric blood for direct matching in cornea transplantation

“…The automated, non-radioactive sequencing was performed as described by Knipper et al [6]. Briefly, the second exon of the HLA-DRB or -DQB gene was amplified using one biotinylated primer.…”

Sequence‐based typing of HLA class II DQB1

“…The strategy presented here couples a low resolution group‐specific PCR with a sequencing endpoint to facilitate simple and accurate analysis. Previously published HLA‐DQB1 SBT methods ( 17–19) use a solid support to denature the PCR product to create single‐stranded template for use in a standard single‐temperature sequencing reaction. We chose cycle sequencing to reduce the number of sample manipulations and because it requires less template.…”