PCR‐RFLP method holds great promise for complete HLA class II genotyping

Inoko, H

doi:10.1111/j.1399-0039.1990.tb01806.x

Cited by 35 publications

(12 citation statements)

References 6 publications

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“…This PCR-RFLP method is more practical and conventional than the PCR-SSO method because digested products can be visualized directly on acrylamide gels, eliminating the need for separate hybridization with multiple probes. However, the original PCR-RFLP method has some problems in that digested fragments located close to each other sometimes obstruct precise analysis of RFLP bands, and some heterozygotes cannot be discriminated from each other (12,13). Precise discrimination of heterozygotes in the DQBl gene is hampered by concomitant amplification of the DQB2 (DXP) gene (14) with the primers previously used (GH28 and GH29), leading to RFLP patterns which were difficult to 53 interpret (11,13).…”

Section: Introductionmentioning

confidence: 99%

“…However, the original PCR-RFLP method has some problems in that digested fragments located close to each other sometimes obstruct precise analysis of RFLP bands, and some heterozygotes cannot be discriminated from each other (12,13). Precise discrimination of heterozygotes in the DQBl gene is hampered by concomitant amplification of the DQB2 (DXP) gene (14) with the primers previously used (GH28 and GH29), leading to RFLP patterns which were difficult to 53 interpret (11,13). To resolve these problems, DQB 1 -specific amplification, particularly groupspecific amplification (12,13), as well as digestion with restriction endonucleases which do not necessitate precise analysis of fragment sizes, will be useful for simple genotyping of DQBl.…”

Section: Introductionmentioning

confidence: 99%

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HLA‐DQB1 genotyping by a modified PCR‐RFLP method combined with group‐specific primers

et al. 1991

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We previously reported a simple technique for HLA-DQB genotyping by digestion of polymerase chain reaction-amplified genes with restriction endonucleases (PCR-RFLP method). However, this method has some problems in that some heterozygotes cannot be discriminated from each other. Furthermore, concomitantly amplified product derived from the DQB2 gene by the primers used previously also obstructs precise DQB1 genotyping. To resolve these problems, we have developed two different pairs of specific primers for selective amplification of the DQB1 gene and also used restriction endonucleases which have either a single cleavage site or, alternatively, no cleavage site in the amplified DNA region, depending on the HLA-DQB1 alleles, making reading of RFLP band patterns much easier. The second exon of the DQB1 gene was selectively amplified by DQw1 group-specific primers and/or DQw2,3,4 group-specific primers using genomic DNAs from 70 HLA-homozygous B-cell lines and 50 healthy Japanese. Of the seven DQw1-associated DQB1 alleles, six alleles could be defined by digestion of 6 restriction enzymes, although DQB1*0602 and DQB1*0603 could not be discriminated from each other because of unavailability of suitable enzymes. Similarly, all of the six DQw2,3,4-associated DQB1 alleles could be defined by digestion of 5 restriction enzymes. Using this modified PCR-RFLP method, complete DQB1 genotyping of all heterozygotes is possible except for discrimination between DQB1*0602 and 0603. Thus this method is simpler and more practical for a routine DNA typing than the PCR-SSO method or our previous PCR-RFLP method.

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Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

HLA‐DQB1 genotyping by a modified PCR‐RFLP method combined with group‐specific primers

et al. 1991

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“…The two sequences differ in codon 35 where TTC encodes Phe in DPBl*SUT instead of the CTC encoding Leu in DPB 1 *0203. The PCR-RFLP method was expected to be able to easily detect a one-base mismatch in HLA class I1 genotyping compared to the PCR-SSO (sequence-specific oligonucleotide) method (9,10). Our results provided the first evidence that the modified PCR-RFLP method for DPBl genotyping can identify the heterozygote which contains an unrecognized allele, even if the unknown allele differs by only one base from a corresponding known one.…”

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confidence: 77%

A new HLA‐DPB1 allele, DPB1SUT (DPB14701)

Koshizaka¹,

Taguchi²,

Ōnishi³

et al. 1994

Tissue Antigens

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“…A non-radioactive "reverse dot blot" method has also been used for SSO typing (17), but the amount and length of oligonucleotide probes must be adjusted and suitable conditions for hybridization determined, which is technically demanding. The PCR-RFLP method is practical and conventional for genotyping, but small fragments or bands located close to each other on the polyacrylamide gels sometimes obstruct precise analysis and some heterozygotes cannot be discriminated from each other (18).In this study we searched for informative restriction enzymes which have a single recognition site in some alleles, but none in other alleles in the amplified segments of the HLA-DQA1 or -DPBl genes (19-26), making reading of the generated RFLP band patterns much simpler. The PCRamplified DNAs digested with these restriction endonucleases (ApaLI, HphI, BsaJI, FokI and MboII for DQA1, Bsp12861, FokI, DdeI, BsaJI, BssHII, Cfrl31, RsaI, EcoNI and AvaII for DPBl) were subjected to electrophoresis.…”

mentioning

confidence: 99%

mentioning

confidence: 99%

Modified PCR‐RFLP method for HLA‐DPB1 and ‐DQA1 genotyping

et al. 1991

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We previously developed a new technique for HLA class II genotyping by digestion of polymerase chain reaction-amplified genes with restriction endonucleases (PCR-RFLP method). This PCR-RFLP method is an efficient and convenient typing technique for class II alleles. However, small fragments or bands located close to each other on polyacrylamide gels sometimes prevent precise analysis of the RFLP bands. Furthermore, the restriction enzymes we have reported in the previous papers are not sufficient to identify the genotypes of all heterozygous individuals. Here, we report an improved PCR-RFLP method using some informative restriction enzymes which have either a single cleavage site or, alternatively, no cleavage site in the amplified DNA region, depending on the HLA alleles, making reading of RFLP band patterns much easier. Each second exon of the HLA-DQA1 or -DPB1 gene was selectively amplified from genomic DNAs of 70 HLA-homozygous B-cell lines and 100 healthy Japanese by PCR. Amplified DNAs were digested with restriction endonucleases and then subjected to electrophoresis assaying simply for cutting, or no cutting, of the DNA. ApaLI, HphI, BsaJI, FokI, MboII and Mn1I can discriminate eight alleles of the DQA1 gene. Similarly 19 alleles of the DPB1 gene can be discriminated with Bsp1286I, FokI, DdeI, BsaJI, BssHII, Cfr13I, RsaI, EcoNI, and AvaII enzymes. This modified PCR-RFLP method can be successfully applied to heterozygotes. Thus, the method is technically simpler and more practical for routine HLA typing work than our previous PCR-RFLP method.

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PCR‐RFLP method holds great promise for complete HLA class II genotyping

Cited by 35 publications

References 6 publications

HLA‐DQB1 genotyping by a modified PCR‐RFLP method combined with group‐specific primers

HLA‐DQB1 genotyping by a modified PCR‐RFLP method combined with group‐specific primers

A new HLA‐DPB1 allele, DPB1SUT (DPB14701)

Modified PCR‐RFLP method for HLA‐DPB1 and ‐DQA1 genotyping

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PCR‐RFLP method holds great promise for complete HLA class II genotyping

Cited by 35 publications

References 6 publications

HLA‐DQB1 genotyping by a modified PCR‐RFLP method combined with group‐specific primers

HLA‐DQB1 genotyping by a modified PCR‐RFLP method combined with group‐specific primers

A new HLA‐DPB1 allele, DPB1*SUT (DPB1*4701)

Modified PCR‐RFLP method for HLA‐DPB1 and ‐DQA1 genotyping

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A new HLA‐DPB1 allele, DPB1SUT (DPB14701)