Sequence-based typing of HLA-A2 alleles using a primer with an extra base mismatch

Ishikawa, Yoshihide; Tokunaga, Katsushi; Kashiwase, Kouichi; Akaza, Tatsuya; Tadokoro, Kenji; Juji, Takeo

doi:10.1016/0198-8859(94)00119-b

Cited by 28 publications

(12 citation statements)

References 9 publications

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“…The difference in nucleotide sequence between the HLA-A null allele (A*O215N) and A*0207 was only a single base substitution in the middle of exon 4. We have previously reported that A'0207 is strongly associated with B46 and DR8.1 (Ishikawa et al 1995), which also form the haplotype carrying the A null allele in the present study. Therefore, this A null allele is most probably generated by a single point mutation of A'0207.…”

Section: Discussionsupporting

confidence: 74%

“…The sequence is shown in Figure 1 with the sequence of A'0207 predicted from the amino acid sequence of the al to a3 domains, because A'0207 has been determined by amino acid sequencing of only these domains (Domenech et al 1988). The nucleotide sequence from exon 2 to exon 3 of A'0207 was confirmed by direct sequencing using HLA-A2-specific primers developed previously (Ishikawa et al 1995). …”

Section: Serological Typingmentioning

confidence: 99%

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HLA-A null allele with a stop codon, HLA-A * 0215N, identified in a homozygous state in a healthy adult

et al. 1995

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Section: Discussionsupporting

confidence: 74%

Section: Serological Typingmentioning

confidence: 99%

HLA-A null allele with a stop codon, HLA-A * 0215N, identified in a homozygous state in a healthy adult

et al. 1995

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“…For the optimization of the assay, we tested primers with and without an extra mismatch. For all three SNPs, we obtained higher specificity when using primers with an extra mismatch (data not shown) as has been previously described (21,22). The alternatives for primer design are limited for this method because the 3 end of the allele-specific primer has to be located directly on the SNP either on the plus or the minus strand of the DNA.…”

Section: Resultssupporting

confidence: 73%

Determination of Allele Frequency in Pooled DNA: Comparison of Three PCR-Based Methods

et al. 2005

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Determination of allele frequency in pooled DNA samples is a powerful and efficient tool for large-scale association studies. In this study, we tested and compared three PCR-based methods for accuracy, reproducibility, cost, and convenience. The methods compared were: (i) real-time PCR with allele-specific primers, (ii) real-time PCR with allele-specific TaqMan probes, and (iii) quantitative sequencing. Allele frequencies of three single nucleotide polymorphisms in three different genes were estimated from pooled DNA. The pools were made of genomic DNA samples from 96 cases with basal cell carcinoma of the skin and 96 healthy controls with known genotypes. In this study, the allele frequency estimation made by real-time PCR with allele-specific primers had the smallest median deviation (MD) from the real allele frequency with 1.12% (absolute percentage points) and was also the cheapest method. However; this method required the most time for optimization and showed the highest variation between replicates (SD = 6.47%). Quantitative sequencing, the simplest method, was found to have intermediate accuracies (MD = 1.44%, SD = 4.2%). Real-time PCR with TaqMan probes, a convenient but very expensive method, had an MD of 1.47% and the lowest variation between replicates (SD = 3.18%).

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“…The match case comprises a correct Watson-Crick base pair at the 3′-primer end, whereas the mismatch case features a non-canonical base pair. The mismatch should result in a less efficient or, ideally, no product amplification[30] [32] [33] [34] [35] [36] [37]. In real-time ASA this is reflected with a delayed or absent fluorescence signal for the mismatch case.…”

Section: Introductionmentioning

confidence: 99%

Variants of a Thermus aquaticus DNA Polymerase with Increased Selectivity for Applications in Allele- and Methylation-Specific Amplification

et al. 2014

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The selectivity of DNA polymerases is crucial for many applications. For example, high discrimination between the extension of matched versus mismatched primer termini is desired for the detection of a single nucleotide variation at a particular locus within the genome. Here we describe the generation of thermostable mutants of the large fragment of Thermus aquaticus DNA polymerase (KlenTaq) with increased mismatch extension selectivity. In contrast to previously reported much less active KlenTaq mutants with mismatch discrimination abilities, many of the herein discovered mutants show conserved wild-type-like high activities. We demonstrate for one mutant containing the single amino acid exchange R660V the suitability for application in allele-specific amplifications directly from whole blood without prior sample purification. Also the suitability of the mutant for methylation specific amplification in the diagnostics of 5-methyl cytosines is demonstrated. Furthermore, the identified mutant supersedes other commercially available enzymes in human leukocyte antigen (HLA) analysis by sequence-specific primed polymerase chain reactions (PCRs).

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Sequence-based typing of HLA-A2 alleles using a primer with an extra base mismatch

Cited by 28 publications

References 9 publications

HLA-A null allele with a stop codon, HLA-A * 0215N, identified in a homozygous state in a healthy adult

HLA-A null allele with a stop codon, HLA-A * 0215N, identified in a homozygous state in a healthy adult

Determination of Allele Frequency in Pooled DNA: Comparison of Three PCR-Based Methods

Variants of a Thermus aquaticus DNA Polymerase with Increased Selectivity for Applications in Allele- and Methylation-Specific Amplification

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