A new method for ABO genotyping using fluorescence melting curve analysis based on peptide nucleic acid probes

Lee, Kyungmyung; Park, Hyun-Chul; An, Sang Hyun; Ahn, Eu-Ree; Lee, Yang-Han; Kim, Mi‐Jung; Lee, Eun-jung; Park, Jae Sin; Jung, Jin Hyuk; Lim, Sung-Soo

doi:10.1016/j.legalmed.2015.04.001

Cited by 5 publications

(2 citation statements)

References 21 publications

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“…The major ABO alleles in the Japanese population are A101, A102, A201, B101, O01, and O02. Among the 13 SNPs residing in exons 6‐7 of the ABO gene, the three SNPs at positions 261, 526, and 803 are enough to identify the A, B, and O alleles which occur frequently in the Japanese population . The droplet‐AS‐PCR method described here was specific for single nucleotide alterations, and utilized primers with a mismatched nucleotide located between the −3 to −1 positions relative to the 3′ terminus of the primer, a feature that was shown to provide high specificity for amplification from the desired templates.…”

Section: Discussionmentioning

confidence: 99%

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Rapid ABO genotyping by high‐speed droplet allele‐specific PCR using crude samples

Taira

Matsuda

Takeichi

et al. 2017

Clinical Laboratory Analysis

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Background ABO genotyping has common tools for personal identification of forensic and transplantation field. We developed a new method based on a droplet allele‐specific PCR (droplet‐AS‐PCR) that enabled rapid PCR amplification. We attempted rapid ABO genotyping using crude DNA isolated from dried blood and buccal cells. Methods We designed allele‐specific primers for three SNPs (at nucleotides 261, 526, and 803) in exons 6 and 7 of the ABO gene. We pretreated dried blood and buccal cells with proteinase K, and obtained crude DNAs without DNA purification. Results Droplet‐AS‐PCR allowed specific amplification of the SNPs at the three loci using crude DNA, with results similar to those for DNA extracted from fresh peripheral blood. The sensitivity of the methods was 5%‐10%. The genotyping of extracted DNA and crude DNA were completed within 8 and 9 minutes, respectively. The genotypes determined by the droplet‐AS‐PCR method were always consistent with those obtained by direct sequencing. Conclusion The droplet‐AS‐PCR method enabled rapid and specific amplification of three SNPs of the ABO gene from crude DNA treated with proteinase K. ABO genotyping by the droplet‐AS‐PCR has the potential to be applied to various fields including a forensic medicine and transplantation medical care.

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Section: Discussionmentioning

confidence: 99%

“…Among these methods, AS‐PCR is superior in terms of specificity/sensitivity and protocols. However, conventional PCR methods require a long reaction time in which almost 1 hour is needed for PCR amplification alone …”

Section: Introductionmentioning

confidence: 99%