Bunce M, Barnardo M C, Welsh K I
Nuffield Dept. of Surgery, Oxford Transplant Centre, Churchill Hospital, UK.
Tissue Antigens. 1998 Aug;52(2):158-74. doi: 10.1111/j.1399-0039.1998.tb02280.x.
An emerging problem of molecular typing methods such as PCR amplification using sequence-specific primers (PCR-SSP) is that they frequently require updating as new alleles are constantly being described which potentially affect the specificity of every PCR-SSP reaction. PCR-SSP uses pairs of primers to detect cis-linked polymorphisms and thus each new allele described must be compared to each individual primer pair. Furthermore, sequence homology between the various loci for class I and class II means that, for example, new HLA-A sequences have to be compared with HLA-B and HLA-C primer mixes to rule out cross-locus amplification. We have developed a computer program known as SSP Manager which is capable of aligning HLA class I and class II sequences obtained from Internet-accessible databases such as GenBank. The program then updates all individual primer specificities held in its database before updating the specificities of all primer mixes. Sets of primer mixes can then be combined from the primer mix directory to create PCR-SSP typing trays which are subsequently analysed by the program. A report is generated which stipulates whether all known sequences are amplified and the reason for apparent failure to test for individual alleles, e.g. a lack of relevant sequence information. SSP Manager has the flexibility to cope with unusual sequences (deletions and insertions), primers with internal mismatches and primers with a deliberate mismatch. The program also has many tools for developing new primer mixes, such as the facility to search for novel reactions using Boolean operators. The organisation and operational use of the SSP Manager program is described and its uses are illustrated with an updated allele list for our previously described Phototyping PCR-SSP class I and class II typing set. The SSP Manager is available on request from the authors.
诸如使用序列特异性引物的PCR扩增（PCR-SSP）等分子分型方法中一个新出现的问题是，由于新的等位基因不断被发现，这些方法常常需要更新，而这可能会影响每个PCR-SSP反应的特异性。PCR-SSP使用引物对来检测顺式连接的多态性，因此，每一个新发现的等位基因都必须与每一对单独的引物进行比对。此外，I类和II类基因座之间的序列同源性意味着，例如，新的HLA-A序列必须与HLA-B和HLA-C引物混合物进行比对，以排除跨基因座扩增。我们开发了一个名为SSP Manager的计算机程序，它能够比对从诸如GenBank等可通过互联网访问的数据库中获取的HLA I类和II类序列。然后，该程序会更新其数据库中所有单独引物的特异性，之后再更新所有引物混合物的特异性。接着，可以从引物混合物目录中组合引物混合物组，以创建PCR-SSP分型板，随后由该程序进行分析。生成的报告会规定是否扩增了所有已知序列以及单个等位基因检测明显失败的原因，例如缺乏相关序列信息。SSP Manager能够灵活处理异常序列（缺失和插入）、内部错配的引物以及有意错配的引物。该程序还有许多用于开发新引物混合物的工具，比如使用布尔运算符搜索新反应的功能。本文描述了SSP Manager程序的组织和操作使用方法，并通过我们之前描述的光分型PCR-SSP I类和II类分型集的更新等位基因列表来说明其用途。作者可应要求提供SSP Manager。
