The oligomers were purchased from Bioneer Inc (Korea). The oligomer sequences were 5′-AAG GCC TGC TGA AAA TGA CTA TGC TCT CGC CAC-3′ (DNA1), 5′-P-TAG CTC CCT TAC CAA TGA CTT CTT TGC ATA TTA CTG GTG CAG GAC C-3′ (DNA2), 5′-GTC ATT GGT AAG GGA GCT AGT GCC GAC ACC AT-3′ (Template DNA), 5′-AAG GCC TGC TGA AAA TGA CT-3′ (Forward primer), and 5′-GGT CCT GCA CCA GTA ATA TGC A-3′ (Reverse primer). PCR premix solution and PNA probe were specially designed from Panagene Corp (Korea). The sequence of the PNA probe was Dab-GGA GCT AGT GGC G-(OEK)-FAM, it contains a dabcyl quencher and a FAM group at the
- terminal sites, respectively. Silver nitrate and co-enzyme (β-NAD) were purchased from Sigma-Aldrich. The thermo-stable DNA ligase (
DNA ligase) was purchased from Bioneer Inc. (Korea), and further puri-fied using the desalting method with an Amicon filter (30-kDa cutoff) to remove metal chelating agents such as EDTA and DDT as well as other interfering chloride ions. The desalted ligase was reassembled with freshly prepared reaction buffer (10x; pH 8.3, 100 mM HEPES, 0.5 M Mg(ClO
, and 0.25 M NaNO
The enzymatic ligation was carried out as follows. Each oligomer DNA1, DNA2, and template DNA (each 1 μM) were mixed with the reaction buffer. Silver ions were added to each reaction solution and the final volume was adjusted to 20 μL with sterilized water to reach a final 1x reaction buffer condition (pH 8.3, 10 mM HEPES, 50 mM Mg(ClO
, and 25 mM NaNO
). The concentrations of silver ions in each tube were 0, 1.5, 3, 6, 9, and 12 μM respectively. The reaction solutions were kept at 50°C on the preheated heat-block for 30 min, and then the ligase (2 μL, 2 U/μL) and coenzyme (2 μL, 0.5 mM) were added to the solution. The ligation reaction was performed at 50 °C for 2 h, and then quenched by cooling the solution to 0 °C. The reaction mixture was diluted and an aliquot was used for qPCR. qPCR was performed using an asymmetric PCR method on Bio-Rad C1000 thermal cycler. The PCR solution consisted of the ligated template DNA (1 μL), forward primer (0.8 μM), reverse primer (0.06 μM), PNA probe (0.8 μM) and PCR premix (10 μL). qPCR was programmed for 15 min at 95 °C, followed by 40 cycles of 15 sec at 95 °C, 30 sec at 56 °C and 15 sec at 72 °C.
Results and Discussion
In our system, the T
value of the mismatched pairs between DNA1 and template DNA is critical to control the ligation reaction. Thus, three Cs mismatched DNA1 was designed to lower sufficiently its T
value. No ligation was expected to occur in absence of silver ions at high temper-ature (50 °C). The binding constant of silver ions to a C-C mismatch base is known to be 10
Thus, 1 μM of each DNA was used for stabilization of the enzymatic ligation with increasing concentration of silver ions. The thermo-stable
DNA ligase was chosen for the ligation reaction under hot temperature. It is active between 45 °C and 65 °C. As expected in control conditions (absence of silver ions), no amplification of the ligated DNA was observed at 50 °C. After addition of silver ions, the oligomers can specifically form C-Ag-C complexes between the DNA1 and template DNA inducing an increase in the T
value of the complexes above 50 °C. Therefore, a stable duplex was formed at 50 °C.
DNA ligase recognized the nicked duplex strands and ligated the nicked duplex to produce the ligated DNA target for subsequent qPCR.
(a) qPCR amplification results, (b) Correlation curve between Ct values and the added molar amounts of silver ions.
As the amount of silver ions in the ligation reaction increased, the quantity of ligated products generated increased. Thus, the amplification signal appeared earlier in qPCR.
presents the qPCR profile of the ligated targets generated using increasing concentration of silver ions in the ligation reaction. In theory, Ct values are correlated with an initial copy number of DNA target. As shown in
, the obtained Ct values are correlated with the concentrations of silver ions (0.06, 0.12, 0.24, 0.36, 0.48 and 0.60 nM) in qPCR.
If silver ions are sequentially added to the C-C mismatch-ed pair then, theoretically, at least 3 equivalents of silver ions are required to form a stable DNA duplex because of the presence of three Cs mismatches in DNA1. However, when just 1.5 equivalents of silver ions were added, a significant amplification of the ligated target was observed. This demonstrates that silver ions were preferentially added to the already silver-bound C-Ag-C complex strand to make DNA duplexes more stable. In other words, the binding of one silver ion to the C-C mismatch facilitates the other silver ion to bind to the adjacent C-C mismatch on the same strand to stabilize the duplex formation. When over 9 equivalents of silver ions were added to the solution, the Ct values were almost saturated, which reflects that the binding constant of C-Ag-C complex was not strong enough to complete the equimolar binding of the Ag
and the C-C mismatches.
The detection limit of silver ions in this system was 1.5 pmol, which corresponds to the 1 μL of the 1.5 μM of silver ion solution used for the ligation reaction. Since the aliquots were diluted, 1000x, the amount of silver ions detectable by qPCR at the femtomolar level. When the ligation reaction was carried out using the full matched sequences of DNA1 in absence of silver ions, a Ct value of 17 was observed indicating that the efficiency of the enzymatic ligation for the C-Ag-C complexes was less than 100%. This may be due to the position of C-Ag-C complex close to the ligation site. To improve the efficiency of the ligation, further studies including adjustment of the sites and the number of C-C mismatches are in progress.
The present work demonstrates a simple two-step strategy for the detection of Ag
ions. This is the first report on the use of an enzymatic ligase technique and qPCR for the detection of Ag
ions. This strategy makes a way to extend the enzymatic alphabet for the detection of metal ions. The detection limit of silver ions in this method was 1.5 pmol in the ligation reaction.
This work was supported by a research grant from the Kongju National University in 2012.
Lai C. Z.
Fierke M. A.
Correa da costa R.
Gladysz J. A.
DOI : 10.1021/ac1013767