A simple and rapid method for the preparation of homologous DNA oligonucleotide hybridization probes from heterologous gene sequences and probes.
We describe a simple and rapid method for the preparation of homologous DNA oligonucleotide probes for hybridization analysis and/or cDNA/genomic library screening. With this method, a synthetic DNA oligonucleotide derived from a known heterologous DNA/RNA/protein sequence is annealed to an RNA preparation containing the gene transcript of interest. Any unpaired 3'-terminal oligonucleotides of the heterologous DNA primer are then removed using the 3' exonuclease activity of the DNA Polymerase I Klenow fragment before primer extension/dideoxynucleotide sequencing of the annealed RNA species with AMV reverse transcriptase. From the determined RNA sequence, a completely homologous DNA oligonucleotide probe is then prepared. This approach has been used to prepare a homologous DNA oligonucleotide probe for the successful library screening of the yeast hybRNA gene starting with a heterologous mouse hybRNA DNA oligonucleotide probe.
Maxwell
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Department of Biochemistry, North Carolina State University, Raleigh 27695-7622.
Sarge
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D000818:Animals; D001483:Base Sequence; D004256:DNA Polymerase I / Q000378:metabolism; D015342:DNA Probes; D005796:Genes; D066298:In Vitro Techniques; D051379:Mice; D009693:Nucleic Acid Hybridization; D012331:RNA, Fungal / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics; D012194:RNA-Directed DNA Polymerase / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics; D012689:Sequence Homology, Nucleic Acid; D013045:Species Specificity
D015342:DNA Probes; D012331:RNA, Fungal; D012333:RNA, Messenger; D012194:RNA-Directed DNA Polymerase; D004256:DNA Polymerase I