Isolation and partial nucleotide sequence of a cDNA clone for human histocompatibility antigen HLA-B by use of an oligodeoxynucleotide primer.

TLDR

The authors generated short, heterogeneous cDNA fragments by reverse‑transcriptase synthesis with a 32P‑labelled oligonucleotide primer and dideoxynucleotides, then isolated the desired HLA‑B clone from a pBR322 cDNA library. They obtained a nearly full‑length HLA‑B7 cDNA clone whose partial sequence matches the antigen’s amino‑acid sequence, showing a highly sensitive approach useful for cloning low‑abundance genes and studying HLA‑region genes and peptide expression.

Abstract

We have isolated a cDNA clone for one of the HLA-B locus alloantigens by hybridization with a 30-nucleotide-long DNA probe. The probe was isolated from a reverse transcriptase (RNA-dependent DNA nucleotidyltransferase)-catalyzed cDNA synthesis reaction on poly(A)-mRNA in which an oligonucleotide (5'-32P)dC-T-T-C-T-C-C-A-C-A-TOH served as a primer and in which dideoxynucleoside triphosphates were used to reduce the size and heterogeneity of the cDNA products. The desired cDNA clone was isolated from a library of recombinant cDNA clones in the plasmid pBR322. The partial nucleotide sequence of the cDNA clone corresponds to the amino acid sequence of HLA-B7 antigen. The approach described in this paper is extremely sensitive and may be useful in cloning other genes for which the corresponding mRNA is present at low levels. This cDNA clone is nearly full length and can be used to isolate and to study the genes within the HLA region and to obtain expression of HLA-B peptides in cells.