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.

Open Access
Publisher Website
Google Scholar
Abstract
The 175-nucleotide-long probes for the DNA of human histocompatibility antigens HLA-DR .alpha. and .beta. were synthesized 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 2-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 c[complementary]DNA 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.