A COMPUTER PROGRAM FOR UPDATING THE SPECIFICITIES OF SEQUENCE-SPECIFIC PRIMER REACTIONS.
          M Bunce, M Barnardo and K Welsh, Oxford Transplant Centre, Oxford, UK.
          PCR-SSP is a widespread method of HLA typing. PCR-SSP systems require updating as new alleles are described which potentially affect the specificity of each PCR-SSP reaction. PCR-SSP uses pairs of primers to detect cis-linked polymorphisms therefore each new allele must be compared to each individual primer pair used. Consequently, checking multiple PCR-SSP reactions by hand is often laborious and error prone. We have developed a computer program, "SSP Manager" which is capable of aligning HLA sequences obtained from Internet databases. SSP Manager then updates all individual primer specificities held in the database before updating the specificities of primer mixes (PM). Reaction sets can then be combined from the PM directory to create typing trays which are analysed by the program creating a report stipulating whether all known sequences are amplified and if individual alleles are apparently not tested for reasons such as a lack of relevant sequence information. SSP Manager copes with sequence deletions and insertions and primers with internal or deliberate mismatches. SSP Manager has facilities for dealing with incomplete allele sequences so that logical assumptions can be made about primers in these regions. The program also has tools for developing new primer mixes such as searching for novel reactions using Boolean operators. To illustrate its uses, SSP Manager program was applied to updating the "Phototyping" PCR-SSP assay. Eleven out of 218 new alleles reported since Phototyping was published were revealed by SSP Manager to be not detected and 7/144 primer mixes contained specificity errors. The errors identified had been a result of clerical mistakes or inappropriate interpretation of the effect of internal mismatches within some primers. We conclude that SSP Manager is a rapid, useful tool for updating and improving SSP typing sets and analysing the integrity of typing sets currently used.