Improvement of Sensitivity in HLA-DRB1 Typing by Semi-Nested PCR-RFLP

Seiichi Inoue, Yuji Yamamoto, Osamu Okamoto, Hiroki Murakami, Satoru Miyaishi, Hideo Ishizu

Research output: Contribution to journalArticlepeer-review

1 Citation (Scopus)

Abstract

A sensitive method of HLA-DRB1 typing was devised using a semi-nested polymerase chain reaction (PCR) followed by a restriction fragment length polymorphism (RFLP) analysis (semi-nested PCR-RFLP method). The first-round amplification (30 cycles) of the semi-nested PCR was performed using DRB generic primer pairs and the second round of PCRs (20 cycles) were performed using DRB1 group-specific primers. The products of the second round PCRs were digested with restriction endonucleases for the typing of HLA-DRB1 alleles. By this method, HLA-DRB1 typing was possible from 10pg of genomic DNA extracted from lymphocytes and from 0.5 μI of 1,000 times diluted blood without DNA extraction. HLA-DRB1 alleles could be typed from a 2-mm long bloodstained cotton thread prepared from 10 times diluted blood and from a 2-mm thread of whole blood bloodstains stored at room temperature for 2 years. From the mixture of blood of two individuals with different genotypes, DRB1 alleles of the minor component were detected down to 1/1,000 of the major component. This semi-nested PCR-RFLP method is useful for HLA-DRB1 typing from extremely small amounts of DNA and from mixed samples.

Original languageEnglish
Pages (from-to)289-296
Number of pages8
JournalActa medica Okayama
Volume52
Issue number6
Publication statusPublished - Dec 1 1998

Keywords

  • HLA-DRB1
  • Polymerase chain reaction
  • Polymorphism
  • Restriction fragment length polymorphism
  • Semi-nested PCR

ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)

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