MHC Protocols pp 191-222 | Cite as

Sequencing Protocols for Detection of HLA Class I Polymorphism

  • Paul P. J. Dunn
  • Steven T. Cox
  • Ann-Margaret Little
Part of the Methods in Molecular Biology™ book series (MIMB, volume 210)


The human leukocyte antigen (HLA) genes located within the human major histocompatibility complex on chromosome 6 are probably the most polymorphic functional genetic loci studied to date. The functional HLA genes encode protein molecules that function in antigen presentation within the immune response. Polymorphism within these genes influences diversity of the immune response against different pathogenic infections. Such polymorphism within the human population is considered an advantage, as diversity in immune responses allows some individuals to be better than others at combating certain infections and thus ensuring survival of the species. However, this polymorphism also serves as a major barrier against the transplantation of human organs and stem cells, where HLA incompatibility between donor and recipient can lead to graft rejection or, in the case of stem cells, graft vs host disease. The study of HLA polymorphism has been led by transplantation biologists because of the implication of HLA matching in improving transplant outcome. HLA polymorphism data is also utilized in anthropological studies, where the frequency of HLA alleles can be used as a marker for analysis of population genetics. As HLA proteins function in the immune response, it is not surprising to find association between different HLA types and immunerelated diseases, such as autoimmune disease, and in this field HLA typing serves as a disease marker in genetic studies.


Polymerase Chain Reaction Polymerase Chain Reaction Product Human Leukocyte Antigen Human Leukocyte Antigen Class Human Leukocyte Antigen Allele 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.


  1. 1.
    Argüello J. R., Little A.-M., Bohan E., Goldman J., Marsh S., and Madrigal J. (1998) High resolution HLA class I typing by reference strand mediated conformation analysis (RSCA). Tissue Antigens 52, 57–66.PubMedCrossRefGoogle Scholar
  2. 2.
    Sanger F., Nicklen S., and Coulson A. (1977) DNA sequencing with chain terminating inhibitors. Proc. Natl. Acad. Sci. USA 74, 5463–5471.PubMedCrossRefGoogle Scholar
  3. 3.
    Mezei L. M. and Storts D. R. (1994) Cloning of PCR products, in PCR Technology: Current Innovations (Griffin H. G. and Griffin A. M., eds.), CRC Press Boca Raton.Google Scholar
  4. 4.
    Clark J. M. (1988) Novel non-templated nucleotide addition reactions catalyzed by procaryotic and eucaryotic DNA polymerases. Nucleic Acids Res. 16, 9677–9686.PubMedCrossRefGoogle Scholar
  5. 5.
    Little A.-M. and Parham P. (1993) HLA class I gene and protein sequence polymorphism, in Histocompatibility Testing: A Practical Approach (Dyer P. and Middleton D., eds.), IRL Press Oxford, pp. 159–190.Google Scholar
  6. 6.
    Hanahan D. (1985) Techniques for transformation of E. coli, in DNA Cloning, Vol 1 (Glover D., ed.) IRL Press Oxford, pp. 109–135.Google Scholar
  7. 7.
    Robinson J., Malik A., Parham P., Bodmer J. G., and Marsh S. G. E. (2000) IMGT/HLA database—a sequence database for the human major histocompatibility complex. Tissue Antigens 55, 280–287.PubMedCrossRefGoogle Scholar
  8. 8.
    Cereb N., Maye P., Lee S., Kong Y., and Yang S.Y. (1995) Locusspecific amplification of HLA class I genes from genomic DNA: locus-specific sequences in the first and third introns of HLA-A,-B, and-C alleles. Tissue Antigens 45, 1–11.PubMedCrossRefGoogle Scholar
  9. 9.
    Cereb N. and Yang S. Y. (1997) Dimorphic primers derived from intron 1 for use in the molecular typing of HLA-B alleles. Tissue Antigens 50, 74–76.PubMedCrossRefGoogle Scholar
  10. 10.
    Ennis P., Zemmour J., Salter R., and Parham P. (1990) Rapid cloning of HLA-A,B cDNA by using the polymerase chain reaction: frequency and nature of errors produced in amplification. Proc. Natl. Acad. Sci. USA 87, 2833–2837.PubMedCrossRefGoogle Scholar
  11. 11.
    Zemmour J., Little A.-M., Schendel D., and Parham P. (1992) The HLA-A,B “negative” mutant cell line C1R expresses a novel HLAB35 allele, which also has a point mutation in the translation initiation codon. J. Immunol. 148, 1941–1948.PubMedGoogle Scholar
  12. 12.
    Little A.-M., Mason A., Marsh S. G. E., and Parham P. (1996) HLA-C typing of eleven Papua New Guineans: identification of an HLA-Cw4/Cw2 hybrid allele. Tissue Antigens 48, 113–117.PubMedCrossRefGoogle Scholar
  13. 13.
    Scheltinga S. A., Johnston-Dow L. A., White C. B., et al. (1997) A generic sequencing based typing approach for the identification of HLA-A diversity. Hum. Immunol. 57, 120–128.PubMedCrossRefGoogle Scholar
  14. 14.
    Dunn P. P., Day S., Harvey J., Fuggle S. V., and Ross J. (1998) Identification of an HLA-C variant allele, Cw*0805, by sequencing based typing. Tissue Antigens 52, 587–589.PubMedCrossRefGoogle Scholar
  15. 15.
    Domena J., Little A.-M., Madrigal J. A., et al. (1993) Structural heterogeneity in HLA-B70, a high-frequency antigen of black populations. Tissue Antigens 42, 509–517.PubMedCrossRefGoogle Scholar
  16. 16.
    Parham P., Arnett K., Adams E., et al. (1997) Episodic evolution and turnover of HLA-B in the indigenous human populations of the Americas. Tissue Antigens 50, 219–232.PubMedCrossRefGoogle Scholar

Copyright information

© Humana Press Inc. 2003

Authors and Affiliations

  • Paul P. J. Dunn
    • 1
  • Steven T. Cox
    • 2
  • Ann-Margaret Little
    • 2
  1. 1.Southmead HospitalBristolUK
  2. 2.Anthony Nolan Research InstituteLondonUK

Personalised recommendations