**4.1.4 Dual-luciferase assay**


Application of Host Cell Reactivation in Evaluating the Effects of Anticancer Drugs

**4.2 HCR for HR repair** 

**4.2.1 Materials** 

and Environmental Toxicants on Cellular DNA Repair Activity in Head and Neck Cancer 473

HR is a reliable mechanism to accurately repair DNA double strand breaks. Here we use PCR to generate two overlapping DNA fragments that contain i) CMV IE promoter and 5' part of *Renilla* luciferase gene, ii) 3'-part of *Renilla* luciferase gene and poly-A tail sequence from pRL-CMV (Fig. 2A) and serve as substrates for HR (Fig. 2B). The two overlapping DNA fragments can also be produced by restriction enzyme digestion and gel elution.

1. Plasmids: pRL-CMV (Promega, Cat. No. E2261) and pCMV-Luc (Liu et al., 2004). 2. PCR primers: RL\_1: 5'-AGA TCT TCA ATA TTG GCC ATT AGC; RL\_2: 5'-TTC TTA TTT ATG GCG ACA TGT TGT; RL\_3: 5'-ACG AGG CCA TGA TAA TGT TGG ACG;

> Reaction mixtures HR\_13 (l) HR\_24 (l) 10PCR buffer 5 5 dNTP (2.5 mM) 4 4 RL\_1 primer (10 M) 2 - RL\_3 primer (10 M) 2 - RL\_2 primer (10 M) - 2 RL\_4 primer (10 M) - 2 ExTaq polymerase (5u/l) 0.25 0.25 Template (40 ng/l) 1 1 DNA/RNAase free- H2O 35.75 35.75 Total volume (l) 50 50

2. Incubate the PCR reaction mixtures at 94°C for 2 min, then run for 30 cycles of amplification (94°C, 45 sec; 55°C, 1 min; 72°C, 1 min) and additional extension step at

3. Purify the PCR products of HR13 fragments (1730 bp, containing CMV IE promoter and 5'-part of *Renilla* luciferase gene) and HR24 fregments (1023 bp, containing 3'-part of *Renilla* luciferase gene and poly-A tail) from 0.8% agarose gels (Fig. 2C) using the Gel-

4. Determine DNA concentration and purity by measuring the absorbance at 260 nm and 280 nm with a UV spectrophotometer. Dilute the HR13-PCR products to 17 ng/l and HR24-PCR products to 10 ng/l with distilled H2O to make the molar ratio of HR13:HR24 = 1:1, by which the same volume of the two DNA fragments can be used

Alternative: the two DNA fragments used for HR can also be generated by using a combination of restriction endonucleases *Bgl*II/*Nhe*I and *Pst*I/*Bam*HI for pRL-CMV (Progema). The use of former restriction enzymes will produce a DNA fragment containing

RL\_4: 5'-CTT ATC GAT TTT ACC ACA TTT GTA.

**4.2.2 Substrate preparation for HR repair** 

1. Set up PCR reaction as below:

72°C for 5 min.

MTM Gel Extraction System kit (Viogene).

for transfection. Store the purified DNA in aliquots at -20°C.

4. Gel-MTM Gel Extraction System (Viogene, Cat No. EG1002).

3. DNA Polymerase: *Ex Taq*™ Polymerase (Takara, Cat No. RR001A).

