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. 1983 Mar;80(6):1531–1535. doi: 10.1073/pnas.80.6.1531

Use of synthetic oligonucleotide probes complementary to genes for human HLA-DR alpha and beta as extension primers for the isolation of 5'-specific genomic clones.

H K Das, P A Biro, S N Cohen, H A Erlich, A von Gabain, S K Lawrance, P G Lemaux, H O McDevitt, B M Peterlin, M F Schulz, A K Sood, S M Weissman
PMCID: PMC393635  PMID: 6403940

Abstract

We have synthesized 175-nucleotide-long probes for the DNA of human histocompatibility antigens HLA-DR alpha and beta by extending on poly(A)+ mRNA from B-cell lines with short synthetic deoxyribonucleotide primers complementary to the predicted nucleotide sequence of the NH2 terminus of both polypeptides. The synthesis of the probe for the alpha-chain DNA was a two-step process starting with 11-mers which were extended by dideoxynucleotide chain termination experiments to a 20-mer of predicted sequence. The synthesized 20-mer was then used to generate a 175-nucleotide cDNA probe which was shown to encode the appropriate amino acids for the alpha chain and was used to select a human genomic DNA clone containing the coding sequences for HLA-DR alpha. For the beta polypeptide an 18-mer homologous to the NH2-terminal sequence of a cDNA clone from another B-cell line was used to extend on poly(A)+ mRNA isolated from a B-cell line. Preliminary sequence analysis of a 175-base-long extension product indicates a match of the cDNA sequence to the published sequence of a clone for HLA-DR beta. Information from these extension experiments helps to establish the sensitivity and specificity of the primer extension method.

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Selected References

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  1. Benton W. D., Davis R. W. Screening lambdagt recombinant clones by hybridization to single plaques in situ. Science. 1977 Apr 8;196(4286):180–182. doi: 10.1126/science.322279. [DOI] [PubMed] [Google Scholar]
  2. Blattner F. R., Williams B. G., Blechl A. E., Denniston-Thompson K., Faber H. E., Furlong L., Grunwald D. J., Kiefer D. O., Moore D. D., Schumm J. W. Charon phages: safer derivatives of bacteriophage lambda for DNA cloning. Science. 1977 Apr 8;196(4286):161–169. doi: 10.1126/science.847462. [DOI] [PubMed] [Google Scholar]
  3. Denhardt D. T. A membrane-filter technique for the detection of complementary DNA. Biochem Biophys Res Commun. 1966 Jun 13;23(5):641–646. doi: 10.1016/0006-291x(66)90447-5. [DOI] [PubMed] [Google Scholar]
  4. Gross-Bellard M., Oudet P., Chambon P. Isolation of high-molecular-weight DNA from mammalian cells. Eur J Biochem. 1973 Jul 2;36(1):32–38. doi: 10.1111/j.1432-1033.1973.tb02881.x. [DOI] [PubMed] [Google Scholar]
  5. Klein J. The major histocompatibility complex of the mouse. Science. 1979 Feb 9;203(4380):516–521. doi: 10.1126/science.104386. [DOI] [PubMed] [Google Scholar]
  6. Korman A. J., Ploegh H. L., Kaufman J. F., Owen M. J., Strominger J. L. Cell-free synthesis and processing of the heavy and light chains of HLA-DR antigens. J Exp Med. 1980 Aug 1;152(2 Pt 2):65s–82s. [PubMed] [Google Scholar]
  7. Kratzin H., Yang C. Y., Götz H., Pauly E., Kölbel S., Egert G., Thinnes F. P., Wernet P., Altevogt P., Hilschmann N. Primärstruktur menschlicher Histokompatibilitätsantigene der Klasse II. 1. Mitteilung: Aminosäuresequenz der N-terminalen 198 Reste der beta-Kette des HLA-Dw2,2;DR2,2-Alloantigens. Hoppe Seylers Z Physiol Chem. 1981 Dec;362(12):1665–1669. [PubMed] [Google Scholar]
  8. Lee J. S., Trowsdale J., Bodmer W. F. cDNA clones coding for the heavy chain of human HLA-DR antigen. Proc Natl Acad Sci U S A. 1982 Jan;79(2):545–549. doi: 10.1073/pnas.79.2.545. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Maniatis T., Hardison R. C., Lacy E., Lauer J., O'Connell C., Quon D., Sim G. K., Efstratiadis A. The isolation of structural genes from libraries of eucaryotic DNA. Cell. 1978 Oct;15(2):687–701. doi: 10.1016/0092-8674(78)90036-3. [DOI] [PubMed] [Google Scholar]
  10. Maxam A. M., Gilbert W. Sequencing end-labeled DNA with base-specific chemical cleavages. Methods Enzymol. 1980;65(1):499–560. doi: 10.1016/s0076-6879(80)65059-9. [DOI] [PubMed] [Google Scholar]
  11. Niederhuber J. E., Frelinger J. A. Expression of Ia antigens on T and B cells and their relationship to immune-response functions. Transplant Rev. 1976;30:101–121. [PubMed] [Google Scholar]
  12. Rosenthal A. S. Determinant selection and macrophage function in genetic control of the immune response. Immunol Rev. 1978;40:136–152. doi: 10.1111/j.1600-065x.1978.tb00404.x. [DOI] [PubMed] [Google Scholar]
  13. Shackelford D. A., Kaufman J. F., Korman A. J., Strominger J. L. HLA-DR antigens: structure, separation of subpopulations, gene cloning and function. Immunol Rev. 1982;66:133–187. doi: 10.1111/j.1600-065x.1982.tb00437.x. [DOI] [PubMed] [Google Scholar]
  14. Smith H. O., Birnstiel M. L. A simple method for DNA restriction site mapping. Nucleic Acids Res. 1976 Sep;3(9):2387–2398. doi: 10.1093/nar/3.9.2387. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Soberon X., Covarrubias L., Bolivar F. Construction and characterization of new cloning vehicles. IV. Deletion derivatives of pBR322 and pBR325. Gene. 1980 May;9(3-4):287–305. doi: 10.1016/0378-1119(90)90328-o. [DOI] [PubMed] [Google Scholar]
  16. Sood A. K., Narang S. A. A rapid and convenient synthesis of poly-thymidylic acid by the modified triester approach. Nucleic Acids Res. 1977 Aug;4(8):2757–2765. doi: 10.1093/nar/4.8.2757. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Sood A. K., Pereira D., Weissman S. M. Isolation and partial nucleotide sequence of a cDNA clone for human histocompatibility antigen HLA-B by use of an oligodeoxynucleotide primer. Proc Natl Acad Sci U S A. 1981 Jan;78(1):616–620. doi: 10.1073/pnas.78.1.616. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Stetler D., Das H., Nunberg J. H., Saiki R., Sheng-Dong R., Mullis K. B., Weissman S. M., Erlich H. A. Isolation of a cDNA clone for the human HLA-DR antigen alpha chain by using a synthetic oligonucleotide as a hybridization probe. Proc Natl Acad Sci U S A. 1982 Oct;79(19):5966–5970. doi: 10.1073/pnas.79.19.5966. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Szostak J. W., Stiles J. I., Tye B. K., Chiu P., Sherman F., Wu R. Hybridization with synthetic oligonucleotides. Methods Enzymol. 1979;68:419–428. doi: 10.1016/0076-6879(79)68031-x. [DOI] [PubMed] [Google Scholar]
  20. Tu C. P., Cohen S. N. 3'-end labeling of DNA with [alpha-32P]cordycepin-5'-triphosphate. Gene. 1980 Jul;10(2):177–183. doi: 10.1016/0378-1119(80)90135-3. [DOI] [PubMed] [Google Scholar]
  21. Wiman K., Larhammar D., Claesson L., Gustafsson K., Schenning L., Bill P., Böhme J., Denaro M., Dobberstein B., Hammerling U. Isolation and identification of a cDNA clone corresponding to an HLA-DR antigen beta chain. Proc Natl Acad Sci U S A. 1982 Mar;79(6):1703–1707. doi: 10.1073/pnas.79.6.1703. [DOI] [PMC free article] [PubMed] [Google Scholar]

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