Isolation of a cDNA clone of the 14-kDa subunit of the signal recognition particle by cross-hybridization of differently primed polymerase chain reactions.
Using an enhancement of the polymerase chain reaction (PCR) technique, we have isolated a complementary DNA encoding SRP14 (14-kDa subunit), one of six proteins contained in the signal recognition particle (SRP). Several pools of degenerate oligonucleotides encoding different peptide sequences of SRP14 were used to generate amplified DNA by the PCR. A cross-hybridization procedure was developed to identify the authentic SRP14 cDNA clone among the amplified DNA products obtained by PCR. The basis of this approach is the assumption that a partial cDNA of SRP14 should be the only DNA product common to two amplification reactions primed with different degenerate oligonucleotide mixtures. The partial canine cDNA of SRP14 identified by this procedure served as a probe to isolate a complete cDNA clone of SRP14 from a mouse embryonic cDNA library in lambda phage gt10.
Strub
K
K
Department of Biochemistry and Biophysics, University of California Medical School, San Francisco 94143-0448.
Walter
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P
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics* / Q000302:isolation & purification; D004259:DNA-Directed DNA Polymerase; D004285:Dogs; D004622:Embryo, Mammalian; D015723:Gene Library; D046911:Macromolecular Substances; D051379:Mice; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009693:Nucleic Acid Hybridization; D015345:Oligonucleotide Probes; D010179:Pancreas / Q000378:metabolism; D016133:Polymerase Chain Reaction / Q000379:methods*; D012261:Ribonucleoproteins / Q000235:genetics* / Q000302:isolation & purification; D018271:Signal Recognition Particle; D019914:Taq Polymerase
D046911:Macromolecular Substances; D015345:Oligonucleotide Probes; D012261:Ribonucleoproteins; D018271:Signal Recognition Particle; C503637:Srp72 protein, mouse; D004247:DNA; D019914:Taq Polymerase; D004259:DNA-Directed DNA Polymerase