TABLE 3.

PCR determination of the relative enrichment of the HSV-1 LAT promoter and DNA polymerase gene regions following ChIP with anti-acetyl histone H3(K9, K14)a

PCR primersSample, no. of cyclesDilutionbFluorescencecIP valued
LATInput, 340.21.29 × 106
0.18.21 × 105
0.057.10 × 105
0.021.19 × 105
IP, 341.10 × 1060.16
Input, 350.21.55 × 106
0.11.18 × 106
0.054.37 × 105
0.025.97 × 105
IP, 351.47 × 1060.18
Input, 360.22.04 × 106
0.11.75 × 106
0.051.27 × 106
0.028.58 × 105
IP, 361.95 × 1060.17
PolInput, 340.27.27 × 105
0.13.79 × 105
0.053.68 × 105
0.021.02 × 105
IP, 342.43 × 1050.044
Input, 350.21.15 × 106
0.19.23 × 105
0.054.65 × 105
0.023.36 × 105
IP, 354.80 × 1050.041
Input, 360.21.34 × 106
0.11.02 × 106
0.058.12 × 105
0.029.40 × 104
IP, 364.46 × 1050.030
  • a Samples included either dilutions of the input (mock-immunoprecipitated sample as described in Materials and Methods) or the actual IP sample [immunoprecipitated with acetyl histone H3 (K9, K14)]. These samples were analyzed by PCR with primers either for the LAT promoter or the HSV-1 DNA polymerase (Pol) promoter. Separate PCR runs were performed for either 34, 35, or 36 cycles.

  • b Input samples were diluted serially as indicated; IP samples were analyzed undiluted.

  • c PCR products were resolved by polyacrylamide gel electrophoresis and stained with SYBR green, and the band intensities were measured on a Storm 860 instrument with ImageQuant software.

  • d The input dilution data were fit by linear regression (Kaleidagraph). The IP fluorescence value was calculated from the linear fit of the input dilution data. The mean values ± standard deviations were 0.17 ± 0.01 for the LAT primers and 0.038 ± 0.007 for the Pol primers.