Phototyping: comprehensive DNA typing for HLA‐A, B, C, DRB1, DRB3, DRB4, DRB5 & DQB1 by PCR with 144 primer mixes utilizing sequence‐specific primers (PCR‐SSP)

Abstract

We have developed a single DNA typing method which uses 144 sequence-specific primer (SSP) reactions to simultaneously detect all known HLA-A, B, C, DRB1, DRB3, DRB4, DRB5 and DQB1 specificities in an allele specific or group specific manner using the same method, reagents, PCR parameters and protocols for all loci. The results from this integrated class I & II method can be visualized on a single photographic or electronic image and hence is described as "Phototyping". Phototyping has an overall resolution greater than or equivalent to good serology and results can be obtained in under 3 hours making the method suitable for genotyping potential cadaver donor peripheral blood without serological backup. This in turn produces the potential for reducing cold ischaemia times in renal transplantation as well as the application of prospective matching to cardiac and liver transplantation. The method has capacity to detect new alleles, for example, novel amplification patterns suggestive of 4 new HLA-B alleles have been detected. The Phototyping set has been used as the sole method of HLA typing for over 1010 individuals. Phototyping is not problem-free; deviations from the standard protocol, poor quality DNA and unsuitable PCR machines can result in individual PCR failures or in incorrect assignment of antigens. Approximately 5% of genotypes were repeated (either partially or fully) because of incomplete or equivocal results.

Variation in immunoregulatory genes determines the clinical phenotype of common variable immunodeficiency

“…Secondly, this method is highly cost-efficient (less than £0.30 per reaction), which may be further enhanced by the incorporation of multiple allele-specific primer pairs in one reaction mixture (PCRmultiplexing). Thirdly, the reaction conditions described in this paper are identical to those previously described for HLA Class I and Class II, 14 and for TNF and lymphotoxin alpha (LT␣), 15 enabling further simplification of disease-association studies. An additional advantage of this approach is that it is particularly suited for the rapid detection of a variety of polymorphisms in a single individual.…”

“…The allele-/haplotype-specific amplicons are smaller than the control amplicons (and thus uppermost) in all reactions except those for ICAM-1 (lanes 31-34) and IL-6 (lanes [7][8], where the reverse is true. Positive lanes: 1 (IL-1␣ −889t homozygous); 3 (IL-1␤ +3962c homozygous); 5 (IL4 −590c homozygous); 7, 8 (IL-6 +3247a/g heterozygous), 9, 11 (IL-10 −1082/−819/−592 a-c-c/g-c-c heterozygous); 13,14,17,18,19 (TGF␤ −800g-−509c/−800g-−509t heterozygous, aa101/P heterozygous and 263T homozygous); 21 allele frequencies is provided in Table 1. All control allele frequencies were in Hardy-Weinberg equilibrium, as determined by 2 × n Chi-square analysis of observed and expected genotype frequencies.…”

“…24 To verify successful amplification, all reaction mixes included control primers for HLA-DRB1 (for amplicons Ͻ600 bp) or human growth hormone (for amplicons Ͼ600 bp). 14,25 Primer sequences and reaction mix composition is listed in Table 6.…”

Expression of inhibitory KIR is confined to CD8+ effector T cells and limits their proliferative capacity

“…HLA-C generic PCR-SSP typing was performed using a combination of 23 sequence-specific primer mixes (Dynal Allset, Compiè gne, France) as described [42]. For TCR expression analysis, CD8 + peripheral T cells stained with mAb GL183 were FACS-sorted.…”

Interleukin-10 gene promoter polymorphism in English and Polish healthy controls. Polymerase chain reaction haplotyping using 3′ mismatches in forward and reverse primers

“…The 12 reactions were developed such that they could be carried out using PCR protocols and parameters identical to those already reported for related cytokines and HLA phototyping. 4,8 The method is simple, robust and cost-effective and may be easily employed in laboratory practice to identify those individuals, who may be predisposed to low or high production of IL-10 protein. It is, however, somewhat limited by the distance between any two polymorphisms.…”