Rapid verification of lambda-cDNA clones using mixed-base oligonucleotide as screening probe and sequencing primer.
A rapid procedure has been developed for the isolation and verification of cDNA clones isolated from a cDNA library based on lambda vectors. Using information of the partial amino acid sequence of a protein, synthetic mixed-base oligonucleotides are first employed as a screening probe using the plaque hybridization procedure. The cDNA inserts of the clones obtained are then directly amplified by polymerase chain reaction (PCR) using primers flanking the cloning site of the vector. Besides being used for cloning into a plasmid vector, the amplified DNA's are also subjected to nucleotide sequence analysis using the same mixed-base oligonucleotides as sequencing primers. This approach allows sequencing through the region of the known amino acid sequence for direct verification of the authenticity of the clones obtained. This procedure has successfully been used for cloning and partial characterization of the gene coding for a platelet aggregation inhibitor.
Au
L C
LC
Department of Medical Research, Veterans General Hospital, Taipei, Taiwan, Republic of China.
Huang
Y B
YB
Teh
G W
GW
Huang
T F
TF
Choo
K B
KB
D000595:Amino Acid Sequence; D010582:Bacteriophage lambda / Q000235:genetics*; D001483:Base Sequence; D004247:DNA / Q000235:genetics / Q000302:isolation & purification*; D015723:Gene Library; D005822:Genetic Vectors; D008969:Molecular Sequence Data; D009710:Nucleotide Mapping; D015345:Oligonucleotide Probes; D016133:Polymerase Chain Reaction
D015345:Oligonucleotide Probes; D004247:DNA