Applications of the Polymerase Chain Reaction to HLA Class II Typing

Bidwell, J. L.

doi:10.1159/000462237

Cited by 2 publications

(2 citation statements)

References 32 publications

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“…HLA matching is essential for kidney and bone marrow transplantation (1)(2)(3)(4) and new molecular biological methods (5) for detection of HLA polymorphism currently provide an opportunity to improve matching of the patients and donors, as well as a research tool to investigate the relationship between HLA disparity and transplant complications. These molecular typing methods include sequence-specific primer (SSP) amplification (6,7), hybridization with sequence-specific oligonucleotide (SSO) probes (8,9), heteroduplex analysis (10)(11)(12), single-strand conformation polymorphism (13,14), and direct nucleotide sequencing (14,15).…”

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confidence: 99%

A novel method for simultaneous high resolution identification of HLA-A, HLA-B, and HLA-Cw alleles.

Argüello

Avakian²,

Goldman³

et al. 1996

Proc. Natl. Acad. Sci. U.S.A.

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We describe a novel high resolution DNA based typing approach for HLA class I alleles, which identifies the recombinational motifs present in exons 2 and 3 of the HLA class I genes. Unique identification patterns for 201 known HLA-A, HLA-B, and HLA-Cw alleles were generated by the use of only 40 probes, which were targeted at these common motifs. The unambiguous identification of the alleles was achieved by the development of a new and powerful allelic separation technique that allows isolation of single alleles after amplification. To validate the method, we have used locus-specific primers to amplify exons 2 and 3 of HLA-A, HLA-B, and HLA-Cw loci from 22 heterozygous and 41 homozygous cell lines. After amplification, the allelic fragments from each locus were separated, blotted, and hybridized with the 40 probes. In all cases, the allelic products could be separated and 81 different class I alleles, 33 HLA-A, 30 HLA-B, and 18 HLA-Cw, were identified according to the predicted probe hybridization patterns.HLA-A, HLA-B, and HLA-Cw antigens are polymorphic cell surface glycoproteins, which are involved in cytotoxic T-cell recognition during the immune response. HLA matching is essential for kidney and bone marrow transplantation (1-4) and new molecular biological methods (5) for detection of HLA polymorphism currently provide an opportunity to improve matching of the patients and donors, as well as a research tool to investigate the relationship between HLA disparity and transplant complications. These molecular typing methods include sequence-specific primer (SSP) amplification (6, 7), hybridization with sequence-specific oligonucleotide (SSO) probes (8, 9), heteroduplex analysis (10-12), single-strand conformation polymorphism (13, 14), and direct nucleotide sequencing (14,15). SSP uses group and/or allele specific sequences for primer design (7) and has been proposed as a single technique for HLA class I and II typing (16); SSO is based on the identification of allele-specific sequences, which are either unique or in certain combinations will allow the identification of the allele (9). The resolution of each method could be increased by using a larger number of primer mixes for SSP and more probes in the case of SSO. These two methods have also been used for class I typing, but their application to high-resolution class I allele analysis has been hindered due to the extensive sequence homology between the HLA-A, -B and -Cw loci (17), between class I classical and nonclassical genes and the reported pseudogenes (18). Direct sequencing is at present used for high-resolution analysis of the class I alleles (14, 15) and, to avoid ambiguous combinations, prior typing is required, which is then followed by allelespecific PCR amplification to generate single allelic fragments. Although this approach achieves the highest resolution, it is expensive and difficult to perform routinely. Heterozygous combinations of certain alleles frequently produce ambiguous

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mentioning

confidence: 99%

A novel method for simultaneous high resolution identification of HLA-A, HLA-B, and HLA-Cw alleles.

Argüello

Avakian²,

Goldman³

et al. 1996

Proc. Natl. Acad. Sci. U.S.A.

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“…RFLP can accurately type the specimen in our hands while PCR SSP did not always yield a positive PCR product for the HLA alleles. This may be related to technical failure of the PCR (17). However, only 4 out of the 96 samples did not yield any PCR product with regard to both the HLA-DRB and -DQB alleles.…”

Section: Dltcussianmentioning

confidence: 98%

Restriction fragment length polymorphism of HLA‐DQ and ‐DR allogenotypes in normal Southern Chinese

Poon

Hawkins

et al. 1993

Tissue Antigens

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We have examined the Taq I restriction fragment length polymorphism (RFLP) of the MHC class II regions of 96 normal Southern Chinese with respect to the HLA-DRB, -DQA, and -DQB genes. The data were compared with the DR and DQ serotyping. RFLP correlates well with the serological typing of the DR types of Chinese. Variants are seen in DR2 and DR12. The variant in DR12 with a 10 kb at the upper band (DR12b) is not found in Caucasoids and is more frequent in Chinese (19.4% of the alleles). With the help of DQA and DQB RFLP the assignment of genotyping of DRB types is facilitated. Some of the linkage disequilibrium patterns among DRB, DQA and DQB are different from Caucasoids. This is particularly obvious in the DR2 and DR5 haplotypes. In the 96 Southern Chinese, the 3 commonest haplotype frequencies are: DR12b, DQ alpha 1b, DQ beta 3b (18.3%); DR9, DQ alpha 3, DQ beta 3a (15.7%); DR4, DQ alpha 3, DQ beta 3a (14.2%). The various DR beta, DQ alpha. DQ beta genes can be accurately defined in Chinese by RFLP. Polymerase chain reactions using sequence-specific primers were performed to confirm the various HLA-DRB and -DQB alleles. The use of RFLP is important in the study of HLA matching and HLA-disease association in Southern Chinese.

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Applications of the Polymerase Chain Reaction to HLA Class II Typing

Cited by 2 publications

References 32 publications

A novel method for simultaneous high resolution identification of HLA-A, HLA-B, and HLA-Cw alleles.

A novel method for simultaneous high resolution identification of HLA-A, HLA-B, and HLA-Cw alleles.

Restriction fragment length polymorphism of HLA‐DQ and ‐DR allogenotypes in normal Southern Chinese

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