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.
Proc Natl Acad Sci U S A
; 80(6): 1531-5, 1983 Mar.
Article
em En
| MEDLINE
| ID: mdl-6403940
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.
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Clonagem Molecular
/
Genes MHC da Classe II
Limite:
Humans
Idioma:
En
Revista:
Proc Natl Acad Sci U S A
Ano de publicação:
1983
Tipo de documento:
Article