Full-length cDNA cloning utilizing the polymerase chain reaction, a degenerate oligonucleotide sequence and a universal mRNA primer.
A full-length cDNA was selectively amplified by the polymerase chain reaction (PCR) utilizing a primer pair consisting of a "universal" 21-base synthetic deoxyoligonucleotide (oligo dT 17GGCC) and a specific degenerate deoxyoligonucleotide sequence (DOS) derived from the N-terminal amino acid sequence. This double-stranded amplified cDNA was uni-directionally cloned into M13mp19 utilizing two restriction sites that had been previously incorporated into the termini of the universal and specific DOS primers. Cloning of the specific cDNA via this PCR amplification with the universal/specific DOS primer pair approach was confirmed by screening with a second DOS contiguous with the DOS employed to prime second (sense)-strand cDNA synthesis. This technique allows for the selective full-length cDNA cloning of low-abundance mRNAs from a single-protein sequence determination.
Cooper
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DL
Department of Pathology, Duke University Medical Center, Durham, NC 27710.
Isola
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N
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular / Q000379:methods*; D004247:DNA / Q000235:genetics*; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides / Q000138:chemical synthesis / Q000235:genetics; D011061:Poly A / Q000235:genetics*; D016133:Polymerase Chain Reaction; D012313:RNA; D012333:RNA, Messenger / Q000235:genetics*; D012334:RNA, Neoplasm / Q000235:genetics
D009838:Oligodeoxyribonucleotides; C034477:RNA primers; D012333:RNA, Messenger; D012334:RNA, Neoplasm; D011061:Poly A; D012313:RNA; D004247:DNA