A strategy to detect and isolate an intron-containing gene in the presence of multiple processed pseudogenes.
We have devised a strategy that utilizes the polymerase chain reaction (PCR) for the detection and isolation of intron-containing genes in the presence of an abundance of processed pseudogenes. The method depends on the genomic DNA sequence between the PCR primers spanning at least one intron in the gene of interest, resulting in the generation of a larger intron-containing PCR product in addition to the smaller PCR product amplified from the intronless pseudogenes. A unique intron probe isolated from the larger PCR product is used for the detection of intron-containing clones from recombinant DNA libraries that also contain pseudogene clones. This method has been used successfully for the selective isolation of an intron-containing rat L19 ribosomal protein gene in the presence of multiple pseudogenes. Analysis of a number of mammalian ribosomal protein multigene families by PCR indicates that they all contain only a single gene with introns.
Department of Eukaryotic Genome Organization and Expression, Imperial Cancer Research Fund, London, United Kingdom.
B
B
Davies
S
S
Feo
E
E
Heard
M
M
Fried
D000818:Animals; D001483:Base Sequence; D015139:Blotting, Southern; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics / Q000302:isolation & purification; D004259:DNA-Directed DNA Polymerase; D005784:Gene Amplification; D005796:Genes; D007438:Introns; D008969:Molecular Sequence Data; D010957:Plasmids; D011544:Pseudogenes; D051381:Rats; D012269:Ribosomal Proteins / Q000235:genetics*; D019914:Taq Polymerase
D012269:Ribosomal Proteins; C050482:Rpl19 protein, rat; D004247:DNA; D019914:Taq Polymerase; D004259:DNA-Directed DNA Polymerase