Aging Aging

322 Middleton, Curran, and Williams(greater specificity and accuracy) of using molecular methods instead of serologicaltyping to define the HLA system have been well shown in transplantationand disease association (2,3). Such approaches should allow future studies,examining whether certain HLA alleles influence longevity, to reach a meaningfulconclusion.Many molecular methods are available to define the HLA alleles. Describedin this chapter is the sequence-specific oligonucleotide probe (SSOP) method.This method is also directly applicable to defining other polymorphic lociwithin the MHC or elsewhere in the genome. The basis of this method is thespecific amplification of the HLA locus by the polymerase chain reaction(PCR) and the subsequent probing of this product by SSOPs. Most of the vastpolymorphism of the HLA system results from conversion events wherebysmall nucleotide sections of one allele (usually no more than 100 bases long)are transferred to another allele. Thus many of the sequences tend to be sharedby alleles and are not allele specific. Therefore probes are used that are sequencespecific. To differentiate the alleles, a battery of probes is required and it is thepattern of reactivity of these probes that distinguishes the HLA alleles.The detection system used in this laboratory consists of labeling the probeswith digoxigenin (DIG) and detecting the hybridization of these probes to acomplementary sequence present in the PCR-amplified HLA allele of an individualby adding an anti-DIG antibody conjugated with alkaline phosphatase(ALP). The ALP then uses disodium 3-(4-methoxyspiro {1, 2-dioxetane –3, 2'-5'-chloro tricyclo [3.3.1.1] decan}-4-yl) phenyl phosphate (CSPD) as its chemiluminescentsubstrate and the light emitted is detected by autoradiography.To define all alleles at any specific locus at the same time would require alarge number of probes as, although each allele group has a specific probepattern, the combined probe pattern of two alleles present in a heterozygousindividual can be identical to the combined probe pattern of another heterozygousindividual with two different alleles. In addition the system would constantlyneed up-dating to take account of newly discovered alleles. In thislaboratory we use a two-tier SSOP system. The first level of resolution isequivalent to very good serology, that is, the allele group is defined, forexample, HLA-A*02. Thereafter, depending on the initial type, a second PCRspecific for a group of alleles is performed and a further set of probes used togive definition to the allele level. Thus the number of probes required is kept toa minimum and, except for exceptional circumstances, only the high-resolutionsystem needs alteration to take account of new alleles. The primers usedfor each locus are listed in Table 1. The primers for HLA-A and -B loci give alocus-specific product covering exons 2 and 3 and the primer for HLA-DRgives a product from exon 2. This product is not specific for the HLA-DRB1locus and amplifies alleles of other HLA-DR loci (e.g., HLA-DRB3 locus).