Rapid screening of libraries with radiolabeled DNA sequences generated by PCR using highly degenerate oligonucleotide mixtures.
The PCR technique can use protein-derived oligonucleotide sequences as primers to develop probes for screening recombinant libraries. Here we report a method with highly degenerate mixtures of oligonucleotides as primers for the PCR that eliminates the need to identify or isolate the DNA sequences derived by PCR. The method uses the pool of PCR-generated DNA sequences radiolabeled during the extension reaction as a probe, combined with highly stringent hybridization and wash conditions that permit only homologous sequences to hybridize and therefore target desired clones. This technique was used successfully to clone the receptor for tumor necrosis factor.
Heller
R A
RA
Institute of Cancer and Developmental Biology, Syntex Research, Palo Alto, CA 94304.
Song
K
K
Freire-Moar
J
J
D000595:Amino Acid Sequence; D001483:Base Sequence; D004247:DNA / Q000032:analysis*; D015342:DNA Probes / Q000138:chemical synthesis*; D015723:Gene Library; D006801:Humans; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D010761:Phosphorus Radioisotopes; D016133:Polymerase Chain Reaction / Q000379:methods*; D011956:Receptors, Cell Surface / Q000737:chemistry / Q000235:genetics*; D018124:Receptors, Tumor Necrosis Factor
D015342:DNA Probes; D009838:Oligodeoxyribonucleotides; D010761:Phosphorus Radioisotopes; D011956:Receptors, Cell Surface; D018124:Receptors, Tumor Necrosis Factor; D004247:DNA