| | FastAP Thermosensitive Alkaline Phosphatase | T4 Polynucleotide Kinase (T4 PNK) |
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| Applications | - Dephosphorylation of DNA, RNA and oligonucleotides
- Dephosphorylation of vector DNA
- Dephosphorylation of proteins
| - Phosphorylation of PCR products
- Phosphorylation of PCR primers and oligonucleotides
- Labeling of sequencing primers
- Detection of DNA modification by the [32P]-postlabeling assay
- Removal of 3’-phosphate group
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| Substrate | - 5’- and 3’-phosphorylated DNA/RNA
- Oligonucleotides
- dNTPs/NTPs
- Phosphorylated proteins
| - ATP*
- DNA/RNA
- Oligonucleotides
- 3’-NMP
- 3’-phosphorylated DNA/RNA
- 3’-P-oligonucleotides
- Deoxynucleoside 3’-monophosphates
- Deoxynucleoside 3’-diphosphates
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| Reaction catalyzed | - Release of phosphate group
| - Transfer of the γ-phosphate from ATP* to the 5’-OH group of DNA/RNA, oligonucleotides, 3’-NMP
- Exchange of phosphate group between 5’-P-oligo-/polynucleotides and ATP (in the presence of ADP)
- Release of 3’-phosphate group
|
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| Reaction products | - 5’, 3’-OH ends of DNA/RNA
- Oligonucleotides
- Deoxynucleosides / nucleosides
- Dephosphorylated proteins
| - 5’-phosphorylated DNA/RNA
- 5’-P-oligonucleotides
- 3’, 5’-NDP
- 3’-dephosphorylated DNA/RNA oligonucleotides
- Deoxynucleosides
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ss – single-stranded
ds – double-stranded
*Although ATP is routinely used as a phosphate donor, CTP, UTP, GTP, dATP, dTTP are equally effective.