CN105385770B - The Dual-ring hairpin probe for detecting bleomycin mediates label-free strand displacement amplification method - Google Patents

Abstract

The invention discloses a double-loop hairpin probe-mediated label-free strand displacement amplification method for detecting bleomycin. When the bleomycin exists, the double-loop hairpin probe breaks at the recognition site and releases a trigger sequence; then, the trigger sequence is combined with the ring part of the signal probe, and under the action of polymerase and nickase, a strand displacement amplification reaction occurs, and finally a large number of G-quadruplex sequences are generated. At the same time, primer strand extension opens the signal probe, exposing the G-quadruplex sequence sequestered in the neck of the signal probe. Finally, the G-quadruplex sequence binds the NMM molecule, generating a fluorescent signal, which is used to quantify the bleomycin. The invention reduces the background signal of the detection system by designing the trigger sequence at the neck of the double-loop hairpin probe; combines the bleomycin cleavage with the strand displacement amplification reaction to realize the sensitive detection of the bleomycin, the detection The limit is 0.34nM. This method has the advantages of simple operation, label-free and good specificity.

Description

Double-loop hairpin probe-mediated label-free strand displacement amplification method for detection of bleomycin

technical field

The invention relates to a detection method for bleomycin, in particular to a double-loop hairpin probe-mediated label-free strand displacement amplification method for detection of bleomycin.

Background technique

Bleomycin is a class of glycopeptide antibiotics secreted by Verticillium streptomycin. Clinically, bleomycin is an important anticancer drug, mainly used in the treatment of various diseases such as squamous cell carcinoma and macrophage tumor (R.H.Blum, S.K.Carter and K.Agre, Cancer, 1973, 31, 903; M. Froudarakis, E. Hatzimichael, L. Kyriazopoulou, K. Lagos, P. Pappas, A. G. Tzakos, V. Karavasilis, D. Daliani, C. Papandreou and E. Briasoulis, Critical Reviews in Oncology/Hematology, 2013, 87, 90; Y.Akiyama, Q.Ma, E.Edgar, A.Laikhter and S.Hecht, Journal of the American Chemical Society, 2008, 130, 9650; R.A.Giroux and S.M.Hecht, Journal of the American Chemical Society, 2010, 132, 16987; Z. Yu, R. Schmaltz, T. Bozeman, R. Paul, M. Rishel, K. Tsosie and S. Hecht, Journal of the American Chemical Society, 2013, 135, 2883.). However, the application of bleomycin to treat a variety of cancers is often accompanied by dose-limiting toxicities, such as nephrotoxicity, pulmonary toxicity, and pulmonary fibrosis (S.Sleijfer, Chest, 2001, 120, 617; J.Hay, S. Shahzeidi and G. Laurent, Archives of Toxicology, 1991, 65, 81.). Therefore, in order to monitor the concentration of bleomycin, reduce toxicity, and obtain the best therapeutic effect, how to construct a detection method for bleomycin has become the main research content of analysts. At present, researchers have established a variety of detection methods for the quantitative analysis of bleomycin, such as high performance liquid chromatography (R.P.Klett, J.P.Chovana and I.H.Danse, Journal of Chromatography:BiomedicalSciences and Applications, 1984,310,361 ; G.K.Shiu and T.J.Goehl, Journal of Chromatography B: Biomedical Sciences and Applications, 1980, 181, 127.), enzyme immunoassay (K.Fujiwara, M.Yasuno and T.Kitagawa, Cancer Research, 1981, 41, 4121) , radioimmunoassay (J.Teale, J.Clough and V.Marks, British Journal of Cancer, 1977, 35, 822; A.Broughton and J.E.Strong, Cancer Research, 1976, 36, 1418.), and microbial analysis (T.Ohnuma, J.F.Holland, H.Masuda, J.A.Waligunda and G.A.Goldberg, Cancer, 33, 1230.) and so on. However, these methods usually have the disadvantages of time-consuming, laborious, harmful to human health and high cost. Therefore, a new, sensitive and specific detection method needs to be established to provide a simpler and more convenient analysis solution for clinical bleomycin concentration monitoring.

It is reported that in the presence of oxygen and metal ions with redox activity, bleomycin can cleave DNA strands by oxidizing deoxynucleotides, and its main recognition sites are 5'-GT-3' and 5'- GC-3'(Q.Ma, Y.Akiyama, Z.D.Xu, K.Konishi and S.M.Hecht, Journal of the American Chemical Society, 2009, 131, 2013; J.Chen, M.K.Ghorai, G.Kenney and J.Stubbe, Nucleic Acid Research, 2008, 36, 3781.). Based on the property that bleomycin cuts DNA strands, researchers designed various DNA molecular probes that recognize bleomycin to construct a simple and sensitive detection method for bleomycin. Currently, these DNA molecular probes are mainly divided into two categories. The first category is single-stranded or hairpin DNA bound or labeled by signaling molecules (B.C.Yin, D.Wu and B.C.Ye, Analytical Chemistry, 2010, 82, 8272; Y.Li, C.C.Huang, J.B.Zheng, H.L.Qi , W.Cao and Y.M.Wei, Biosensors and Bioeletronics, 2013, 44, 177; Y.F.Qin, Y.F.Ma, X.Jin, L.L.Zhang, G.J.Ye and S.L.Zhao, Analytica Chimica Acta, 2015, 866, 84; F.Li , Y. Feng, C. Zhao, P. Li and B. Tang, Chemical Communications, 2012, 48, 127.). After this type of probe interacts with bleomycin, it can release signal molecules and generate corresponding signal responses. The second category is a functional neck-loop DNA structure with a trigger sequence (F.L.Gao, J.P.Lei and H.X.Ju, Chemical Communications, 2013, 49, 7561.). After this type of probe interacts with bleomycin, it can release the trigger sequence and trigger the signal amplification reaction, expressing a cleavage event as multiple repeated signal generation events. Therefore, this kind of DNA molecular probe is more conducive to improving the signal intensity and sensitivity of the detection method. Gao et al. designed a functional hairpin probe with two 5'-GC-3' recognition sites in the neck and a trigger sequence in the loop for highly sensitive detection of bleomycin (F.L.Gao, J.P.Lei and H.X.Ju, Chemical Communications, 2013, 49, 7561.). However, since the trigger sequence is located in the loop of the probe, non-specific hybridization between the uncleaved DNA molecular probe and the subsequent reaction chain will cause relatively high background signals and false positive signals, which limits the application of this probe.

Contents of the invention

The object of the present invention is to solve the above problems and provide a double-loop hairpin probe-mediated label-free strand displacement amplification method for detecting bleomycin.

In order to achieve the above object, the present invention adopts the following technical solutions:

A double-ring hairpin probe that recognizes and detects bleomycin, the double-ring hairpin probe includes: two loops, a recognition site and a trigger site, the two loops are: having a DNA arm The terminal loop and the neck convex loop with two DNA arms, the recognition site is located at the neck of the double-loop hairpin, the trigger sequence is used to trigger the subsequent amplification reaction, and the bases in the trigger sequence participate in the formation of DNA arms and neck loops of a double loop hairpin probe.

A double-ring hairpin probe-mediated label-free strand displacement amplification method for detecting bleomycin. When the target bleomycin exists, the double-ring hairpin probe breaks at the recognition site and releases the trigger sequence Then, the trigger sequence is combined with the ring portion of the signal probe, and under the action of the polymerase and the nickase, a strand displacement amplification reaction occurs to generate a large amount of fluorescent dye binding sequences. At the same time, the primer chain extension will The signal probe is opened, and the fluorescent dye-binding sequence sealed in the neck of the signal probe is exposed. Finally, the fluorescent dye-binding sequence binds to the fluorescent molecule to generate a fluorescent signal, and the bleomycin is quantified by the detected fluorescent signal; The double loop hairpin probe is as described in claim 1.

Preferably: the fluorescent dye binding sequence at the neck of the signal probe is a G-quadruplex sequence, and the fluorescent molecule is NMM.

Preferably: the recognition site is 5'-GTGC-3'.

Preferably: the trigger sequence is 5'-GAGGAAGAAGAGAGGGAAGGA-3' (as shown in SEQ ID No: 1).

Preferably: the sequence of the double-loop hairpin probe is TCC TTC CCT CTC AAA ACC TCG CAC CAA AAGGTG C GA GGA AGA AGA GAG GGA AGGA (as shown in SEQ ID No: 2).

The sequence of the fluorescent probe is CCC AAC CCG CCC TAC CCT TTT T GA TCC TTC CCT CTC TTCTTC CTC CCT CAA AAA GGG TAG GGC GGG TTG GG (as shown in SEQ ID No: 3).

Preferably: the specific steps of the strand displacement amplification reaction are: take 1 μL 2mM dNTPs, 1U KF polymerase, 4UNt.AlwI, 3 μL 0.75 μM SP and 2 μL 10×NEB buffer2 (10mM Tris-HCl, 10mM MgCl ₂ , 50mMNaCl, 1.0mM dithiothreitol, pH 7.9) was added to the above reaction system, and reacted at 37°C for 40 minutes.

Preferably: in the amplification method, the bicyclic hairpin probe is 100 nM, the signal probe is 75 nM, the DNA polymerase is 1 U, the nicking endonuclease is 4 U, and the NMM concentration is 6 μM.

A test kit for detecting bleomycin, comprising a double-ring hairpin probe and a signal probe, the double-ring hairpin probe as described in claim 1; the signal probe is a hairpin structure, and the signal The neck of the probe has a fluorescent dye binding sequence; the trigger sequence of the double-loop hairpin probe is paired with the loop sequence of the signal probe, fluorescent molecules, DNA polymerase, nicking endonuclease, 10×NEB buffer2 (10mM Tris-HCl, 10 mM MgCl ₂ , 50 mM NaCl, 1.0 mM dithiothreitol, pH 7.9).

Beneficial effects of the present invention:

The invention constructs a double-circle hairpin probe for the recognition and detection of bleomycin. The double loop hairpin probe mainly contains two regions: bleomycin 5'-GTGC-3' recognition site and trigger sequence. In addition, a double-loop hairpin probe has two loop portions, and the loop with two DNA arms is called a neck loop. Different from the functional hairpin probes in the prior art, the trigger sequence of the double-loop hairpin probe is sealed in the neck of the probe in a semi-closed mode, effectively avoiding non-specific hybridization. Semi-closed mode, that is, most of the bases in the trigger sequence participate in forming the DNA arms on both sides of the neck convex loop, and only a small part of the bases constitute the neck convex loop (such as the triggering of the double-ring hairpin probe used in the embodiments of the present invention). The sequence is: 5'-GAGG AAGAA GAGAGGGAAGGA-3', only the underlined part participates in the neck ring, and the rest constitute the DNA arms on both sides). This design pattern has two prominent advantages. On the one hand, the bicyclic hairpin probe uses intramolecular hybridization to silence the trigger sequence, effectively avoiding non-specific hybridization. On the other hand, after the cleavage reaction of the double-loop hairpin probe, a double helix structure with a convex loop in the middle is generated; since the convex loop reduces the stability of the double helix structure, the trigger sequence unwinds with its incomplete complementary strand , and then generate a stable double helix with a sufficient number of bases and subsequent reaction chains, and finally produce an obvious positive signal. Based on the double-loop hairpin probe, combined with the strand displacement amplification (SDA) reaction, the present invention constructs a novel fluorescent sensing platform for the detection of bleomycin. In this method, a large number of DNA single strands are generated by strand displacement amplification reaction and the G-quadruplex/NMM complex emits fluorescence, and the label-free detection of bleomycin based on amplification technology is realized for the first time. Binding of NMM dye molecules by single-stranded DNA products and open signaling probes generates enhanced fluorescent signals, and the method exhibits superior sensitivity. The detection limit of the method is 0.34nM, and the method has the advantages of simple operation, label-free and good specificity. In addition, a human serum recovery experiment was carried out in the experiment, and the results showed that the detection method has great potential for clinical application.

Description of drawings

Figure 1 is the experimental schematic diagram of the BLM detection system based on the bicyclic recognition probe and strand displacement amplification reaction. A. Bleomycin cuts the bicyclic hairpin probe, releases the trigger sequence, and detects the signal under the action of polymerase. The needle is opened to expose a G-quadruplex sequence, B. After the trigger sequence is combined with the signal probe, the strand displacement amplification reaction is triggered under the action of polymerase and nickase;

Figure 2 (A) is polyacrylamide gel electrophoresis verification of bleomycin cleavage and strand displacement amplification amplification reaction; M: DNA ladder; (1) BHP; (2) SP; (3) BHP+BLM; ( 4) BHP+BLM+SP; (5) BHP+BLM+SP+KF polymerase; (6) BHP+BLM+SP+KF polymerase+Nt.AlwI; (7) BHP+SP+KF polymerase+Nt.AlwI; (B) Fluorescence emission spectrum of detection system: Curve a, BHP+SP+KF polymerase+Nt.AlwI+NMM; Curve b, BHP+BLM+SP+KF polymerase+Nt.AlwI+NMM;

Fig. 3 (A) Fluorescence spectra under different bleomycin concentrations: the curve a → h is sequentially 0nM, 2nM, 5nM, 20nM, 100nM, 150nM, 200nM, 220nM; (B) the relationship between fluorescence response and bleomycin concentration The linear relationship between;

Figure 4 shows the effect of different targets on the fluorescence response of the detection system.

Detailed ways

The present invention will be further described below in conjunction with the accompanying drawings and embodiments.

Materials and Instruments Biloop hairpin probe (BHP) and signal probe (SP) oligonucleotide sequences were synthesized and purified by Sangon Biotechnology Co., Ltd. (Shanghai, China). Bleomycin A5, mitomycin and daunorubicin were purchased from China National Institutes for Food and Drug Control. Actinomycin D was purchased from Melone Pharmaceutical Co., Ltd. (Dalian, China). Ferrous chloride (FeCl ₂ ^● H ₂ O) was purchased from Tianjin Guangfu Institute of Fine Chemicals. Klenow fragment (3'-5'exo-) polymerase and Nt.AlwI were purchased from New England Biolabs Ltd. (Beijing, China). dNTPs were purchased from Thermo Fisher Scientific Ltd. (China). NMM was purchased from Frontier Scientific Inc. (Utah, USA). Clinical serum samples were obtained from the Affiliated Hospital of Shandong University. All other chemical reagents were of analytical grade. All aqueous solutions used in the experiment were prepared with ultrapure water (resistivity>18.25MΩ ^● cm).

15 mM Phosphate Buffer: 13.4 mM Na ₂ HPO ₄ , 1.6 mM NaH ₂ PO ₄ and 50 mM NaCl (pH=8.0).

Fluorescence spectra were obtained by scanning at room temperature using a fluorometer Hitachi F-2500 (Japan). The excitation wavelength is 399nM, and the scanning range is 550nM-680nM. Both excitation and emission slit widths were 10 nM. The real-time voltage is 700V.

Bleomycin activation and bicyclic hairpin probe cleavage reaction: first, the bicyclic hairpin probe and the signal probe were annealed at 95°C for 5 minutes, cooled slowly to room temperature, and set aside; then, the bleomycin and chloroform Mix equal concentrations of iron and incubate at room temperature for 30 minutes to obtain activated bleomycin; finally, mix the above-mentioned bleomycin and double-ring hairpin probe thoroughly, and incubate at 37°C for 30 minutes to make bleomycin Fully cleaves double loop hairpin probes.

Strand displacement amplification reaction: After the cleavage reaction is completed, take 1μL 2mM dNTPs, 1U KF polymerase, 4UNt.AlwI, 3μL 0.75μM SP and 2μL 10×NEB buffer2 (10mM Tris-HCl, 10mM MgCl ₂ , 50mMNaCl, 1.0mM dithiothreitol, pH 7.9) was added to the above reaction system, and reacted at 37°C for 40 minutes.

Fluorescence measurement: After the strand displacement amplification reaction was completed, 1.2 μL of 150 μM NMM and 4.8 μL of 1M KCl were added to the reaction system, and incubated at 37° C. for 30 minutes. Take 30 μL of the solution obtained from the final reaction and place it in a fluorescent cell, and perform spectral scanning according to the scanning parameters described in the Materials and Instruments section.

Gel electrophoresis: 10% polyacrylamide gel electrophoresis to verify the bicyclic hairpin probe cleavage reaction and strand displacement amplification reaction. In order to clearly observe the DNA bands, we used high concentrations of bleomycin, double-ring hairpin probes and signal probes for experiments. Take 10 μL of the solution obtained from the final reaction, mix well with 2 μL of 6×loading buffer, load the sample, and run the gel. Finally, the obtained DNA gel was stained with ethidium bromide (EB) for 5 minutes, rinsed with water, and imaged under an ultraviolet imaging system.

Results and discussion

Experimental principle

As shown in Figure 1, first, bleomycin cleaves the double-loop hairpin probe at the recognition site to generate a new short hairpin structure and a double helix with a collar in the middle. Since the neck loop reduces the stability of the double helix structure, the double helix unwinds and releases a DNA sequence, which is named Cut-primer (CP for short). Then, the cleavage primer strand binds to the ring portion of the signal probe, and under the action of KF polymerase, the signal probe is used as the template strand to extend to form a double helix structure with the recognition site of nickase Nt.AlwI . Next, the nicking enzyme nicks at the recognition site and creates a new polymerase binding site. Thus, polymerase extension, replication strand release, and nicking enzyme nicking process cycle, and finally generate a large number of single-stranded G-quadruplex sequences; at the same time, primer strand extension opens the signal probe, exposing G-quadruplex sequence at the neck of the needle. Finally, the G-quadruplex sequence binds the NMM, producing a detectable fluorescent signal.

Feasibility verification

In order to verify whether the bicyclic hairpin probe can function according to the above design process and the feasibility of the analysis scheme, we conducted a 10% polyacrylamide gel electrophoresis experiment. As shown in Figure 2A, compared with lane 1, a darker DNA band appeared in lane 3 at the same position, and a new DNA band appeared at a position close to 20 bp. This indicated that the cleavage reaction of the bicyclic hairpin probe occurred. When the signal probe was added to the reaction solution of bleomycin and bicyclic hairpin probe, a new DNA band with a migration speed significantly slower than that of the signal probe appeared in lane 4. This new DNA band indicates that after the cleavage reaction, the CP strand is released and combines with the SP loop to form a hybrid double helix structure. After adding KF polymerase, a bright DNA band appeared at the position of 60 bp in lane 5, indicating that the CP chain took the SP chain as a template to undergo a chain extension reaction under the action of the polymerase to generate a more stable DNA double helix. When KF polymerase and nicking enzyme were added to the reaction system at the same time, a DNA band with a migration speed faster than 20 bp appeared in lane 6, indicating that the SDA reaction produced a large number of single-stranded DNA molecules. Lane 7 has only the DNA bands of the double-loop hairpin probe and the signal probe, indicating that the double-helix structure of the double-loop hairpin probe has sufficient stability, and no SDA reaction has occurred in the system. In order to further prove the feasibility of the double-ring hairpin probe design and analysis scheme, we carried out corresponding fluorescence spectrum scanning experiments. As shown in Figure 2B, the detection system exhibited a low background signal (Curve a) in the absence of the target. This indicates that the CP sequence in the bicyclic hairpin probe cannot be used to generate a signal in the strand displacement amplification reaction when no cleavage reaction occurs. When the target bleomycin was added to the reaction system, the detection system showed a significantly improved response signal (Curve b). This indicated that after the cleavage reaction, the CP sequence was released and used in the subsequent strand displacement amplification reaction to generate an enhanced fluorescent signal. The above results show that the detection scheme is feasible, and the double-loop hairpin probe can successfully silence the trigger chain, thereby reducing or eliminating the background signal brought by non-specific hybridization in homogeneous analysis.

Experimental condition optimization

In order to obtain the best sensing performance, parameters such as the concentration of the double-loop hairpin probe, the concentration of the signal probe, the concentration of the enzyme and the concentration of NMM were optimized in the experiment. Finally, 100 nM of the double-loop hairpin probe, 75 nM of the signal probe, 1 U of KF polymerase, 4 U of Nt.AlwI, and 6 μM of NMM were selected.

Linearity and Range

Under the optimal experimental conditions, we investigated the sensitivity and linear range of the detection system. As shown in Figure 3, when the concentration of bleomycin gradually increased from 0 nM to 220 nM, the fluorescence response intensity also increased. The fluorescence intensity was linear with the concentration of bleomycin in the range of 0nM-220nM (R=0.998). Detection limit 0.34nM.

specificity

In order to examine the specificity of the method, bleomycin, mitomycin, daunorubicin and actinomycin D were selected as targets for investigation. The results are shown in Figure 4. Under the same experimental conditions, the detection system only showed a strong response signal to bleomycin, indicating that the detection method has good specificity.

Recovery inspection

In order to investigate the practical application ability of this method, we conducted a human serum recovery experiment. The experimental results are between 95%-100%, indicating that the method has potential clinical application.

Summarize

Based on the double-loop hairpin probe-mediated strand displacement amplification reaction, this work constructed a label-free fluorescent strategy for the sensitive detection of bleomycin. To the best of our knowledge, this method enables the label-free detection of bleomycin based on DNA amplification technology for the first time. The limit of detection was 0.34 nM. The recovery rate of human serum meets the requirements, indicating that the method has potential practical application ability in drug analysis and clinical application.

Although the specific implementation of the present invention has been described above in conjunction with the accompanying drawings, it does not limit the protection scope of the present invention. Those skilled in the art should understand that on the basis of the technical solution of the present invention, those skilled in the art do not need to pay creative work Various modifications or variations that can be made are still within the protection scope of the present invention.

Claims (9)

1. a kind of Dual-ring hairpin probe of detection bleomycin mediates label-free strand displacement amplification method, it is characterized in that：Work as target In the presence of object bleomycin, Dual-ring hairpin probe is broken at recognition site, and discharges trigger sequence；Then, sequence is triggered Row are combined with the ring portion of signal probe, and under the action of polymerase and nicking enzyme, and strand displacement amplification reaction occurs, and are generated big The fluorescent dye binding sequence of amount, at the same time, primer chain extension opens signal probe, the fluorescence that signal probe neck is sealed up for safekeeping Dyestuff binding sequence is exposed, and finally, fluorescent dye binding sequence binds fluorescent molecular, generates fluorescence signal, passes through detection To fluorescence signal bleomycin is quantified；The Dual-ring hairpin probe includes：Two ring portions, recognition site and trigger bit Point, described two ring portions are：Tool is there are one the end-rings of DNA arms and has the neck bulge loop there are two DNA arms, the recognition site position Base in the neck of Dual-ring hairpin, the trigger sequence participates in constituting the neck and neck bulge loop of Dual-ring hairpin probe.

2. the method as described in claim 1, it is characterized in that：The fluorescence binding sequence of the signal probe neck is tetra- serobilas of G- Sequence, the fluorescent molecular are NMM.

3. the method as described in claim 1, it is characterized in that：The recognition site is 5 '-GTGC-3 '.

4. the method as described in claim 1, it is characterized in that：The trigger sequence such as SEQ ID No：Shown in 1.

5. the method as described in claim 1, it is characterized in that：The sequence of the Dual-ring hairpin probe such as SEQ ID No：Shown in 2.

6. the method as described in claim 1, it is characterized in that：The sequence of fluorescence probe such as SEQ ID No：Shown in 3.

7. the method as described in claim 1, it is characterized in that：Strand displacement amplification reaction the specific steps are：Take 1 μ L, 2 mM DNTPs, 1 U KF polymerases, 4 U Nt.AlwI, 3 0.75 μM of μ L SP and 2 μ 10 × NEB of L buffer2, described 10 × NEB buffer2：10 mM Tris-HCl, 10 mM MgCl₂, 50 mM NaCl, 1.0 mM dithiothreitol, pH 7.9；Above-mentioned reaction system is added, is reacted 40 minutes at 37 DEG C, you can.

8. the method as described in claim 1, it is characterized in that：In the amplification method, Dual-ring hairpin probe is 100 nM, signal Probe is 75 nM, and archaeal dna polymerase 1U, nicking restriction endonuclease is 4U, a concentration of 6 μM of NMM.

9. a kind of kit of detection bleomycin, it is characterized in that：Including Dual-ring hairpin probe and signal probe, the bicyclic hair Pressing from both sides probe includes：Two ring portions, recognition site and triggering site, described two ring portions are：Tool there are one DNA arms end-rings and There are two the neck bulge loop of DNA arms, the recognition sites to be located at the neck of Dual-ring hairpin for tool, and the base in the trigger sequence participates in Constitute the neck and neck bulge loop of Dual-ring hairpin probe；The signal probe is hairpin structure, and the neck of the signal probe has Fluorescent dye binding sequence；The trigger sequence of the Dual-ring hairpin probe and the ring portion sequence of signal probe are matched, fluorescent molecular, Archaeal dna polymerase, nicking restriction endonuclease, 10 × NEB buffer2,10 × NEB buffer2：10 mM Tris-HCl, 10 mM MgCl₂, 50 mM NaCl, 1.0 mM dithiothreitol, pH 7.9.