Laboratories participating in the Anthropology/Human Diversity Project have the option of using either the standard SSOP dot blot system being developed by the Technology Development Core or an immobilized probe array/reverse lineblot system developed by Dr. Erlich's laboratory at Roche Molecular Systems. In the reverse lineblot format, the labeled PCR product is hybridized in a single reaction to an array of SSO probes immobilized on a nylon membrane. Approximately 56 probes are used for high resolution HLA-A, 85 probes for high resolution HLA-B and 35 probes for high resolution HLA-C typing. This typing method will be made available to laboratories participating in the Human Diversity project.
Reverse Lineblot Technology
The reverse lineblot system utilizes an immobilized probe array in which oligonucleotide probes are deposited in a linear array on a nylon membrane using an IVEK instrument. The PCR product is generated using biotinylated primers. The presence of biotinylated PCR product bound to a specific probe is detected using streptavidin-horseradish peroxidase (HRP) and a chromogenic, soluble substrate to produce a blue "line" at the position of the positive probe. Genotyping software can interpret the probe reactivity pattern as a genotype and indicates potential ambiguities, but the raw probe reactivity data for both dot blot and reverse line blot will be submitted to the central database. The current typing systems use a separate primer pair for the second and the third exons of the class I loci and use 56 probes for HLA-A, 85 probes for HLA-B, and 35 probes for HLA-C.
Quality Control
Quality Control procedures established for the standard SSOP typing system for detection of class I and class II polymorphisms will be used for the reverse lineblot system as well. Reverse lineblot reagents that have been approved by the Technology Development Core will be sent to participating laboratories. The performance of the participating laboratories using these reagents will be examined by the results obtained with the IHWG QC panel of cell lines to be distributed through the QC Laboratory (Jennifer Ng). Laboratories achieving at least 92% accuracy for each locus typed will be approved for the data submission phase of the workshop. Additional quality control samples will be provided by the QC laboratory throughout the workshop to assist laboratories and project leaders in evaluating the quality of ongoing typing.
Availability of Reagents
Reverse lineblot reagents are available now!
Order form (pdf download)
Typing Protocol
13th IHWG HLA Class I Reverse Line Strip Typing Protocol
Hit Tables (Excel)
HLA-A
HLA-B
HLA-C
RLS Software Update
November, 2000
RLS HLA-C Probe 14
