A safe alternative to radiolabeling of oligonucleotide probes.

The North2South Random Prime Labeling Kit is based on the procedure of Feinberg and Vogelstein(Ref.4,5). Random heptanucleotides containing all possible sequences are annealed to a denatured DNA template. These act as primers for complementary strand synthesis by DNA Polymerase (Klenow fragment, 3'-5' exo-). Biotinylated dNTPs in the reaction mix are incorporated into the newly synthesized DNA. This protocol yields biotin-labeled DNA of high activity for use as probes in hybridization experiments such as Southern and Northern hybridizations. The resulting hybrids can be detected with streptavidin-horseradish peroxidase (HRP) and a chemiluminescent substrate kit such as North2South Chemiluminescent Hybridization and Detection Kit.

Highlights:

• Works with as little as 100 ng starting DNA template
• Exonuclease-free Klenow fragment in the DNA labeling kit produces higher yields
• Each reaction yields probe sufficient for approximately three (10 x 10 cm) blots

References:

1. Hirsch, J. and Henry, S. (1986). Expression of the Saccharomyces cerevisiae inositol-1-phosphate synthase (INO1) gene is regulated by factors that affect phospholipid synthesis. Mol. Cell. Biol. 6, 3320-3328.
2. Hoffman, C. and Winston, F. (1987). A ten-minute DNA preparation from yeast efficiently releases autonomous plasmids for transformation of Escherichia coli. Gene 57, 267-272.
3. Sambrook, J., Fritsch, E. and Maniatis, T. (eds.). Molecular Cloning, 2nd Ed. (1989). Cold Spring Harbor Press. Plainview, New York. (Product # 17946)
4. Feinberg, A.P. and Vogelstein, B. (1983). Anal. Biochem. 132, 6.
5. Feinberg, A.P. and Vogelstein, B. (1983). Anal. Biochem. 137, 266.