Isolation of a cDNA clone for the human HLA-DR antigen α chain by using a synthetic oligonucleotide as a hybridization probe

D. Stetler, Hriday Das, J. H. Nunberg, R. Saiki, R. Sheng-Dong, K. B. Mullis, S. M. Weissman, H. A. Erlich

Research output: Contribution to journalArticle

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Abstract

We have used a synthetic 20-nucleotide hybridization probe to isolate a cDNA clone encoding the α chain of the HLA-DR antigen from a cDNA library constructed from membrane-bound poly(A) + mRNA. A set of synthetic 11-nucleotide fragments, potentially complementary to the codons for amino acids 11-14 of the HLA-DR α chain, were used to prime a cDNA synthesis reaction on various poly(A) + mRNA templates. Extension of the primers in the presence of a single dideoxynucleotide triphosphate resulted in an 18-nucleotide cDNA product whose sequence corresponded to the NH 2-terminal amino acids of the HLA-DR α chain. An oligonucleotide was synthesized based on this sequence information and its specificity for HLA-DR α mRNA was confirmed by primer extension and blot analysis. The CDNA library made from mRNA from the lymphoblastoid cell line CA-SC was probed with 32P-labeled cDNA synthesized on poly(A) + mRNA from a B-cell line (CA-SC) or from a T-cell line (Molt-4) to enrich for B-cell-specific clones. A set of CDNA clones that hybridized preferentially with the B-cell probe was screened with the 32P-labeled 20-nucleotide probe. The cDNA clone isolated by this procedure is 1,100 nucleotides long; the nucleotide sequence of the 5' end of the cDNA insert corresponds to the amino acid sequence of the HLA-DR α chain. Hybridization of this cDNA clone to genomic blots suggests that the HLA-DR α chain is encoded by a single-copy gene. One of the restriction endonucleases used in genomic DNA digests reveals a restriction fragment polymorphism.

Original languageEnglish
Pages (from-to)5966-5970
Number of pages5
JournalProceedings of the National Academy of Sciences of the United States of America
Volume79
Issue number19 I
DOIs
StatePublished - 1 Dec 1982

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HLA-DR Antigens
Oligonucleotides
Complementary DNA
Clone Cells
Nucleotides
Messenger RNA
B-Lymphocytes
Cell Line
Dideoxynucleotides
Amino Acids
DNA Restriction Enzymes
Gene Library
Codon
Libraries
Amino Acid Sequence
T-Lymphocytes
Membranes
DNA
Genes

Cite this

Stetler, D. ; Das, Hriday ; 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 α chain by using a synthetic oligonucleotide as a hybridization probe. In: Proceedings of the National Academy of Sciences of the United States of America. 1982 ; Vol. 79, No. 19 I. pp. 5966-5970.
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abstract = "We have used a synthetic 20-nucleotide hybridization probe to isolate a cDNA clone encoding the α chain of the HLA-DR antigen from a cDNA library constructed from membrane-bound poly(A) + mRNA. A set of synthetic 11-nucleotide fragments, potentially complementary to the codons for amino acids 11-14 of the HLA-DR α chain, were used to prime a cDNA synthesis reaction on various poly(A) + mRNA templates. Extension of the primers in the presence of a single dideoxynucleotide triphosphate resulted in an 18-nucleotide cDNA product whose sequence corresponded to the NH 2-terminal amino acids of the HLA-DR α chain. An oligonucleotide was synthesized based on this sequence information and its specificity for HLA-DR α mRNA was confirmed by primer extension and blot analysis. The CDNA library made from mRNA from the lymphoblastoid cell line CA-SC was probed with 32P-labeled cDNA synthesized on poly(A) + mRNA from a B-cell line (CA-SC) or from a T-cell line (Molt-4) to enrich for B-cell-specific clones. A set of CDNA clones that hybridized preferentially with the B-cell probe was screened with the 32P-labeled 20-nucleotide probe. The cDNA clone isolated by this procedure is 1,100 nucleotides long; the nucleotide sequence of the 5' end of the cDNA insert corresponds to the amino acid sequence of the HLA-DR α chain. Hybridization of this cDNA clone to genomic blots suggests that the HLA-DR α chain is encoded by a single-copy gene. One of the restriction endonucleases used in genomic DNA digests reveals a restriction fragment polymorphism.",
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Isolation of a cDNA clone for the human HLA-DR antigen α chain by using a synthetic oligonucleotide as a hybridization probe. / Stetler, D.; Das, Hriday; Nunberg, J. H.; Saiki, R.; Sheng-Dong, R.; Mullis, K. B.; Weissman, S. M.; Erlich, H. A.

In: Proceedings of the National Academy of Sciences of the United States of America, Vol. 79, No. 19 I, 01.12.1982, p. 5966-5970.

Research output: Contribution to journalArticle

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T1 - Isolation of a cDNA clone for the human HLA-DR antigen α chain by using a synthetic oligonucleotide as a hybridization probe

AU - Stetler, D.

AU - Das, Hriday

AU - Nunberg, J. H.

AU - Saiki, R.

AU - Sheng-Dong, R.

AU - Mullis, K. B.

AU - Weissman, S. M.

AU - Erlich, H. A.

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N2 - We have used a synthetic 20-nucleotide hybridization probe to isolate a cDNA clone encoding the α chain of the HLA-DR antigen from a cDNA library constructed from membrane-bound poly(A) + mRNA. A set of synthetic 11-nucleotide fragments, potentially complementary to the codons for amino acids 11-14 of the HLA-DR α chain, were used to prime a cDNA synthesis reaction on various poly(A) + mRNA templates. Extension of the primers in the presence of a single dideoxynucleotide triphosphate resulted in an 18-nucleotide cDNA product whose sequence corresponded to the NH 2-terminal amino acids of the HLA-DR α chain. An oligonucleotide was synthesized based on this sequence information and its specificity for HLA-DR α mRNA was confirmed by primer extension and blot analysis. The CDNA library made from mRNA from the lymphoblastoid cell line CA-SC was probed with 32P-labeled cDNA synthesized on poly(A) + mRNA from a B-cell line (CA-SC) or from a T-cell line (Molt-4) to enrich for B-cell-specific clones. A set of CDNA clones that hybridized preferentially with the B-cell probe was screened with the 32P-labeled 20-nucleotide probe. The cDNA clone isolated by this procedure is 1,100 nucleotides long; the nucleotide sequence of the 5' end of the cDNA insert corresponds to the amino acid sequence of the HLA-DR α chain. Hybridization of this cDNA clone to genomic blots suggests that the HLA-DR α chain is encoded by a single-copy gene. One of the restriction endonucleases used in genomic DNA digests reveals a restriction fragment polymorphism.

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