Nick Translation and Random Hexamer Labeling of DNA
- 503 Downloads
Labeled nucleotides (radioactive or fluorescent) can be incorporated efficiently into double-stranded DNA by nick translation. Nick translation works by using DNase and DNA polymerase I enzymes. DNase cuts one strand of the DNA, exposing 5′-phosphoryl and 3′-hydroxyl (OH) termini DNA. Polymerase I adds deoxyribonucleoside triphosphate (dNTPs), including labeled dNTPs to the exposed 3′-OH strand and at the same time, the polymerase exonuclease activity digests from the exposed 5′ end. In this way, a new complementary strand, including labeled dNTPs is produced (1). It is also possible to incorporate radioactive nucleotides into DNA using a enzymatic primer extension technique (2). In this method, random hexanucleotides are annealed to the denatured DNA to be used as the probe. These are used as a primer for enzymatic extension in the presence of the four dNTPs, one of which is radiolabeled.
KeywordsAmersham Pharmacia Biotech Stop Solution Complementary Strand Nick Translation Unincorporated Nucleotide