From a short amino acidic sequence to the complete gene.
A useful strategy directed to the isolation of a required gene with a high GC content is reported. Using a degenerate oligonucleotide probe, deduced from the amino terminus of a protein, it is possible to obtain a fragment of DNA containing its encoding gene by PCR amplification. Furthermore, the cloning of a desired gene can be accomplished in two steps by using an oligonucleotide deduced (i) from an internal sequence, (ii) from a consensus sequence, or (iii) from a DNA sequence adjacent to a disrupting element (transposon, insertion sequence, cassette). This method, which could be applied to a bacteriophage, plasmid, or cosmid genomic library, has been successfully used for cloning several genes from different biological systems.
Miñambres
B
B
Departamento de Bioquímica y Biología Molecular, Universidad de León, Spain.
Olivera
E R
ER
García
B
B
Naharro
G
G
Luengo
J M
JM
D000595:Amino Acid Sequence / Q000235:genetics*; D001419:Bacteria / Q000235:genetics; D001482:Base Composition; D003001:Cloning, Molecular / Q000379:methods*; D016384:Consensus Sequence / Q000235:genetics; D017124:Conserved Sequence / Q000235:genetics; D017931:DNA Primers / Q000235:genetics; D005796:Genes / Q000235:genetics*; D005822:Genetic Vectors; D015698:Genomic Library; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016254:Mutagenesis, Insertional / Q000235:genetics; D015345:Oligonucleotide Probes / Q000235:genetics; D009842:Oligopeptides / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016133:Polymerase Chain Reaction
D017931:DNA Primers; D015345:Oligonucleotide Probes; D009842:Oligopeptides; D010446:Peptide Fragments