| Assay Method Information | |
| | Biochemical Assay |
| Description: | Human POLγ was incubated with a linear, partially double-stranded template containing an 85 base pair double-stranded DNA stretch and a 15 nucleotides long single-stranded 5′-tail (hereafter referred to as the “exonuclease template”). The exonuclease template showed high fluorescence intensity when incubated with the DNA intercalator SYBR Green I. SYBR Green I is a double-stranded DNA-binding dye, which can be used to quantify double-stranded DNA. When bound to double-stranded DNA, SYBR Green I fluorescence increases by up to 100-fold. In the absence of deoxynucleotide triphosphates (dNTPs), the exonuclease activity of POLγ degrades the 85-mer strand, which leads to the formation of single-stranded DNA (ssDNA) and decreased fluorescent intensity. The assay was performed in a 384-well plate format. Addition of compounds that inhibit POLγ exonuclease activity resulted in a higher fluorescent signal, due to reduced degradation of the 85-mer oligonucleotide. |
| Affinity data for this assay | |
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