Jove
Visualize
Contact Us
JoVE
x logofacebook logolinkedin logoyoutube logo
ABOUT JoVE
OverviewLeadershipBlogJoVE Help Center
AUTHORS
Publishing ProcessEditorial BoardScope & PoliciesPeer ReviewFAQSubmit
LIBRARIANS
TestimonialsSubscriptionsAccessResourcesLibrary Advisory BoardFAQ
RESEARCH
JoVE JournalMethods CollectionsJoVE Encyclopedia of ExperimentsArchive
EDUCATION
JoVE CoreJoVE BusinessJoVE Science EducationJoVE Lab ManualFaculty Resource CenterFaculty Site
Terms & Conditions of Use
Privacy Policy
Policies

Related Experiment Videos

Fluorotyping of HLA-A by sequence-specific priming and fluorogenic probing

K Slateva1, M A Camps, R Blasczyk

  • 1Department of Transfusion Medicine, Hannover Medical School, Germany.

Tissue Antigens
|December 24, 1998
PubMed
Summary

We developed a novel fluorotyping method for HLA-A typing, improving automation and throughput compared to traditional PCR-SSP. This fluorescence-based assay offers quantitative results and aids in identifying new alleles.

Related Concept Videos

You might also read

Related Articles

Articles linked to this work by shared authors, journal, and citation graph.

Sort by
Same author

The microstructure in the placenta is influenced by the functional diversity of HLA-G allelic variants.

Immunogenetics·2019
Same author

Granulocyte colony-stimulating factor impairs CD8(+) T cell functionality by interfering with central activation elements.

Clinical and experimental immunology·2016
Same author

Determination of unacceptable HLA antigen mismatches in kidney transplant recipients: recommendations of the German Society for Immunogenetics.

Tissue antigens·2015
Same author

Lentivirus-induced 'Smart' dendritic cells: Pharmacodynamics and GMP-compliant production for immunotherapy against TRP2-positive melanoma.

Gene therapy·2015
Same author

A future with less HLA: potential clinical applications of HLA-universal cells.

Tissue antigens·2015
Same author

Modulation of heme oxygenase-1 by metalloporphyrins increases anti-viral T cell responses.

Clinical and experimental immunology·2014

Area of Science:

  • Immunogenetics
  • Molecular Biology
  • Biotechnology

Background:

  • Conventional sequence-specific primed PCR (PCR-SSP) for HLA-A typing faces limitations in sample throughput, automation potential, and contamination risks.
  • Agarose gel electrophoresis used in PCR-SSP is time-consuming and labor-intensive.

Purpose of the Study:

  • To develop and validate a fluorotyping strategy for high-resolution HLA-A typing.
  • To overcome the limitations of conventional PCR-SSP, including low throughput and automation challenges.

Main Methods:

  • Developed a fluorescence-based PCR-SSP assay utilizing target-specific, dual-labeled fluorogenic probes (FAM/TAMRA).
  • Probes hybridized to a conserved region in the 2nd intron of HLA-A alleles, enabling detection via Taq DNA polymerase exonuclease activity.

Related Experiment Videos

  • Utilized 24 individual PCR primer mixes for HLA-A specificity and a separate probe for an internal control amplicon.

    • Main Results:

    • The fluorotyping assay demonstrated simultaneous and differential detection of HLA-A specific products and an internal control.
    • Quantitative fluorescence data provided by the assay can resolve ambiguities in heterozygous vs. homozygous genotype discrimination.
    • The method shows potential for detecting novel HLA-A alleles not covered by existing primer mixes.

    Conclusions:

    • Fluorotyping offers a more automated, higher-throughput, and quantitative alternative to conventional PCR-SSP for HLA-A typing.
    • The quantitative nature of fluorotyping aids in resolving complex genotyping scenarios and discovering new alleles.