1. Nucleic acid labeling Radioactive deoxynucleoside triphosphate (dNTP); labeled with H 3 (tritium) or P 32.dNTP Purposes: 1. keep tracking small amounts of nucleic acids in cloning experiments. 2. Production of radioactive probes (probe = single pure sequence) to be used in hybridization experiments.

2. End labeling Dephosphorylation using phosphatase. Polynucleotide kinase transfers a radiolabeled phosphate groups from an ATP source onto 5’ ends. The product is a labeled DNA with low specific activity.

3. Nick translation DNA polymerase I (DNA pol I) repairs a nick found naturally, or caused by low concentration of DNase I (nuclease). DNA pol I (nuclease, polymerase) catalyses a strand replacement reaction putting radioactive dNTPs. The result is a highly labeled DNA molecule.

4. Primer extension DNA is denatured by heating. Primer (six nucleotides) annealed to the single stranded DNA (ssDNA). The Klenow of DNA pol I (devoid of nuclease activity) synthesizes a copy of the template strand putting radioactive dNTP. The product is a DNA of very high specific activity.