Shortened protocol for terminal deoxynucleotidyl transferase-dependent PCR.

Several methods have been developed for determining the sequence specificity of DNA damaging agents in single-copy genes in mammalian cells (10). The ligation-mediated PCR (LMPCR) technique can be used for those compounds that cleave DNA and produce a 5′-phosphate (9,14). For those compounds that form stable adducts with DNA (e.g., cisplatin), there are three different techniques that can be used: linear amplification (15), singlestrand ligation PCR (sslig-PCR) (6), and terminal deoxynucleotidyl transferase-dependent PCR (TDPCR) (3,7, 13). The linear amplification technique is not sufficiently sensitive and is not widely used. The sslig-PCR technique was found to be unreliable and involved binding to streptavidin beads. The more recently developed TDPCR technique has been found to be very sensitive, reliable, and does not involve streptavidin beads. The drawback of the TDPCR technique is the long and complicated protocol as originally reported. In this paper, we describe a shortened protocol for TDPCR that reduces the time from four or five days to two days without significantly affecting the efficiency or accuracy of the process. The original paper described the use of TDPCR to determine DNA-adduct formation by UV light. In this paper, we show that the TDPCR technique can also be used to investigate cisplatin damage in a single-copy human gene. Cisplatin is a cancer chemotherapeutic agent that forms adducts with DNA (1,8). Cisplatin does not cleave DNA but forms covalent intrastrand adducts with purine bases in DNA—mainly via the N-7 of guanine (1,5). The cisplatin adducts inhibit the passage of DNA polymerase (10) and hence can be detected by the TDPCR procedure. The human epsilon-globin gene was used as the target DNA sequence (17). Cisplatin-modified genomic human DNA was prepared as previously described (11,12). The TDPCR procedure used in this study was a shortened protocol derived from the method of Komura and Riggs (7). Genomic DNA (approximately 1 μg) was linearly amplified in a 10-μL reaction containing (final composition) 16.6 mM (NH4)2 S O4, 6.7 mM MgCl2, 67 mM Tris-HCl, pH 8.8, 300 μM each dATP, dGTP, dTTP, dCTP, 0.87 U AmpliTaq DNA polymerase (Applied Biosystems, Foster City, CA, USA), and 10 pmol primer 5′-AAATGGAAATTTGTGTTGCAGATAG-3′ [specific to the human epsilon-globin promoter (17)]. There were 25 temperature cycles of 30 s at 95°C, 60°C for 60 s, and 72°C for 90 s, followed by a 5-min incubation at 72°C. After thermal cycling, 2 μL of the linear amplification reaction were added to an 18-μL solution that gave a final composition of 10 U terminal deoxynucleotidyl transferase (TdT) (Life Technologies, Rockville, MD, USA), 1× buffer supplied by the manufacturer, and 2 mM rGTP (Sigma, St. Louis, MO, USA). The sample was then incubated at 37°C for 15 min. The mixture volume was made up to 100 μL with water, and the DNA was precipitated by the addition of 10 μL 3 M sodium acetate and 250 μL 100% ethanol. The precipitate was dissolved in 10 μL TE (1 mM Tris-HCl, pH 7.5, 0.1 mM EDTA). For the ligation reaction, a 2. 4μL volume was transferred to a new tube that contained 3.6 μL ligation mixture, giving a final composition (in 6 μL) of 1× One-Phor-All Buffer PLUS (Amersham Pharmacia Biotech, Melbourne, Australia), 1 mM ATP, 0.2 U T4 DNA ligase (Amersham Pharmacia Biotech), and 10 pmol unidirectional linker. The linker used was linker γ from Komura and Riggs (7) prepared by annealing two oligonucleotides with the sequences 5′-GCGGTGACCCGGGAGATCTGAATTCCC-3′and 5′AATTCAGATCTCCCGGGTCACCGC3′. After incubation at 17°C for 16 h, samples were overlaid with mineral oil, incubated at 95°C for 5 min, and 4 μL, containing 166 nmol (NH4)2S O4, 670 nmol Tris-HCl, pH 8.8, 67 nmol MgCl2, 3 nmol each dATP, dGTP, dTTP, dCTP, 0.87 U AmpliTaq DNA polymerase, 1 pmol 5′-[32P]-labeled nested primer of sequence 5′-AGGAGCCAACAAAAAAGAGCTCAG3′ [specific to the human epsilon-globin promoter (17)], and 3 pmol upper strand of linker α (7) (5′-GCGGTGACCCGGGAGATCTGAATTC-3′) were added. (It is important that the nested primer is [32P]-end-labeled in the absence of dithiothreitol.) The reaction was then subjected to the same thermal cycle conditions as for the linear amplification. Two microliters of the reaction products were then mixed with 2 μL loading dye containing 80% formamide, 50 mM NaOH, 1 mM EDTA, 0.1% bromophenol blue, and 0.1% xylene cyanol before being electrophoresed on a 6% (w/v) polyacrylamide DNA sequencing gel and scanned using a PhosphorImager (Amersham Pharmacia Biotech). The main aim of this research was to establish a shortened protocol for TDPCR. The original TDPCR protocol (7) had the following order of 12 steps: linear amplification with a gene-specific oligonucleotide, ethanol precipitation 1, terminal transferase ribo-G tailing, ethanol precipitation 2, ligation of linker oligonucleotides, PCR with a nested gene-specific oligonucleotide, phenol extraction, ethanol precipitation 3, electrophoresis, electroblotting, hybridization with a third gene-specific oligonucleotide, and autoradiography. We first wished to replace the laborintensive final steps of phenol extraction, ethanol precipitation 3, electroblotting, hybridization with a third gene-specific oligonucleotide, and autoradiography. These steps were replaced by PCR with a 32P-labeled nested gene-specific oligonucleotide, electrophoresis, and autoradiography. This latter modification is commonly used for LMPCR (2,16). Using this modified protocol, cisplatin damage could be detected (Figure 1, lanes 7 and 8), and the procedure was shown to work efficiently. The preferred sites of cisplatin damage were at runs of consecutive guanines as found previously (10). It is not possible to directly compare the original TDPCR procedure and the modified protocol on the same gel because the original TDPCR procedure used a blotting technique to visualize the bands, whereas, in the modified protocol, we directly label the DNA before gel electrophoresis. However, we have compared the modiBenchmarks