Use of Synthetic Oligonucleotide Probes Complementary to Genes for Human HLA-DR α and β as Extension Primers for the Isolation of 5′-Specific Genomic Clones

• Published in: Proceedings of the National Academy of Sciences - PNAS 1983-03, Vol.80 (6), p.1531-1535
• Main Authors: Das, Hriday K., Biro, P. Andrew, Cohen, Stanley N., Erlich, Henry A., Von Gabain, Alexander, Lawrance, Simon K., Lemaux, Peggy G., McDevitt, Hugh O., Peterlin, B. Matija, Schulz, Marie-Francoise, Sood, Ashwani K., Weissman, Sherman M.
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Amino acids; B lymphocytes; Cell lines; Cloning, Molecular - methods; Complementary DNA; DNA; DNA - genetics; Exons; Genes, MHC Class II; Genomics; HLA-DR Antigens; Humans; Messenger RNA; Nucleotides; Oligodeoxyribonucleotides; RNA, Messenger - genetics; Sequence analysis
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.80.6.1531

We have synthesized 175-nucleotide-long probes for the DNA of human histocompatibility antigens HLA-DR α and β by extending on poly(A)+mRNA from B-cell lines with short synthetic deoxyribonucleotide primers complementary to the predicted nucleotide sequence of the NH2terminus of both polypeptides. The synthesis of the probe for the α -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 α -chain and was used to select a human genomic DNA clone containing the coding sequences for HLA-DR α . For the β 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 β . Information from these extension experiments helps to establish the sensitivity and specificity of the primer extension method.