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.
|Number of pages||6|
|Journal||Biochemical and Biophysical Research Communications|
|State||Published - Nov 30 1988|
Bibliographical noteFunding Information:
ACKNOWLEDGMENTS: We would like to thank Robert Hamatake and Craig Giroux for making the yeast genomic library available to us. The technical assistance of Joyce Liu in isolating the yeast hybRNA gene clone from this lambda gtll library is also gratefully acknowledged. This work was supported by PHS grant CA38015 (E.S.M.). Contribution from the Department of Biochemistry, College of Agriculture and Life Sciences and College of Physical and Mathematical Sciences. Paper No. 11785 of the Journal Series of the North Carolina Agricultural Research Service, Raleigh, North Carolina 27695-7643.
ASJC Scopus subject areas
- Molecular Biology
- Cell Biology