- Email: [email protected]
Optimization of the masking molecule for active-site-protected immobilization of Taq DNA polymerase and its application
Analytical Biochemistry 432 (2013) 139–141
Contents lists available at SciVerse ScienceDirect
Analytical Biochemistry journal homepage: www.elsevier.com/locate/yabio
Notes & Tips
Optimization of the masking molecule for active-site-protected immobilization of Taq DNA polymerase and its application Gumjung Lim a, Joungmok Kim b, Jeong Hee Kim b,⇑ a b
Department of Chemistry, Kyung Hee University, Seoul 130-701, Republic of Korea Department of Oral Biochemistry and Molecular Biology, Kyung Hee University, Seoul 130-701, Republic of Korea
a r t i c l e
i n f o
Article history: Received 11 July 2012 Received in revised form 19 September 2012 Accepted 24 September 2012 Available online 1 October 2012 Keywords: Active-site protection Taq DNA polymerase Masking molecule Immobilization
a b s t r a c t The method of oriented and activity-preserved immobilization of biologically active proteins based on concepts of active-site masking and kinetic control was further developed in this study. Minimal requirements for the masking DNA molecule were found to be a 50 overhang of 5–7 nucleotides and a doublestranded region of 11–13 bp to retain approximately 70% of the enzyme activity. The amplification range of protected immobilized (PIM) Taq DNA polymerase was over 1.2 kb. These data suggest that PIM Taq DNA polymerase can be used for various commercial applications. Ó 2012 Elsevier Inc. All rights reserved.
We previously reported a new effective immobilization method that involves active-site protection and kinetic control of immobilization density using Taq DNA polymerase as a model system [1]. Taq DNA polymerase is an enzyme whose substrate is much larger than those of other enzymes. The immobilization of such enzymes on a solid surface with preserved enzyme activity has been a challenging subject, while that of enzymes with smaller substrates such as glucose has been more readily achieved [2,3]. In the new immobilization method, the active site of the enzyme is protected with a masking molecule, and the masking molecule preferentially facilitates covalent coupling of the enzyme to the surface at a location away from the active site without relying upon a site-specific immobilization scheme. In this study, the masking DNA molecule for protection of the active site of Taq DNA polymerase during the immobilization process was further optimized to explore application possibilities of the active-site-protected immobilized (PIM) Taq DNA polymerase to the commercial level and the results are reported. Materials and methods Chemicals and reagents The following chemicals were purchased and used for the immobilization reaction: (D,L)-thioctic acid (Aldrich, USA), ⇑ Corresponding author. Fax: +82 2 961 0915. E-mail address: [email protected] (J.H. Kim). 0003-2697/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.ab.2012.09.033
1-ethyl-3-[3-(dimethylamino)propyl]carbamide (Sigma, USA), and N-hydroxysuccinimide (Aldrich). Chemicals used for the selfassembled monolayer (SAM) fabrication, 12-mercaptododecanoic acid [HS(CH2)11COOH] and 6-mercapto-1-hexanol [HS(CH2)6OH], were purchased from Aldrich. Au-coated slides were purchased from EMF Corp. (USA). Taq DNA polymerase and deoxynucleotide mixture were from Applied Biosystems (USA) and Takara (Japan), respectively. Primers were synthesized from Bioneer (Korea). Other chemicals were purchased from Sigma or from other common sources. Immobilization of active-site-protected Taq DNA polymerase and solid-phase polymerase chain reaction (PCR) The Au substrate used was a glass plate 3.0 5.0 mm in size on which Au was vacuum-deposited to about 1000 Å thickness. The fabrication of SAM on the Au surface and immobilization of Taq DNA polymerase were performed as reported previously [1], except that the masking DNA molecule was changed to DNA molecules with various lengths and structures as described below. PCR was performed as described in Lim et al. [1]. A 65-base single-stranded DNA (65-mer, single-stranded DNA; 30 -CCAGCTGCCATAGCTATTTTCTTTTCTTTCTTAAGTTCTTTTCTTTTCCTAGGTGATCAAGATCT-50 , 25 fmol) was used as a template, and KS and SK primers (50 -CGAGGTCGACGGTATCG-30 and 50 -TCTAGAACTAGTGGATC-30 , 10 pmol each) were used for PCR in a reaction volume of 50 ll. The temperature cycle was set as follows: hot start step, 94 °C, 10 min; 35 cycles of 94 °C, 30 s, 50 °C, 60 s, 72 °C, 30 s, unless
140
Notes & Tips / Anal. Biochem. 432 (2013) 139–141
specified otherwise. For masking molecule optimization experiments, 35 cycles of PCR were performed: 94 °C, 30 s, 42 °C, 30 s, 72 °C, 30 s. For larger amplicons, pBluescriptII KS(+) was used as a template and primer sets used were as specified in the figure legend. PCR cycling conditions were as follows: hot start step, 94 °C, 10 min; 35 cycles of 94 °C, 30 s, 50 °C, 30 s, 72 °C, 2 min. Final extension was performed at 72 °C for 10 min. A part of the PCR solution was sampled after the PCR and analyzed by agarose gel electrophoresis. The PCR products were visualized by fluorescence from ethidium bromide staining and quantified with a densitometer (Ultra-Lum Imaging system, USA). All experiments were performed at least in triplicate. Results and discussion Optimization of masking molecule for active-site protection of Taq DNA polymerase To prepare an immobilization matrix, a SAM of x-functionalized thiols on a Au surface was formed as described previously [1]. The active site of the target enzyme, Taq DNA polymerase, was protected by masking with a partially double-stranded DNA molecule and then immobilized through amide bonds to the activated SAM. The amount of the Taq DNA polymerase used (0.75 pmol) corresponded to the amount that can form three monolayers on the area of 3 5 mm of the Au substrate. First, the minimum required size of the noncomplementary single-stranded region of the masking DNA molecule for protecting the active site of Taq DNA polymerase was determined. The size of a single-stranded DNA was fixed as a 17-mer by using the KS primer, and the size of the complementary single-stranded DNA was varied from a 16- to a 65-mer. Sequences of these DNA molecules Size of 5’ overhang of masking DNA
a
Relative Enzyme Activity
b
M -1
0 1
2 3
5
7
9
51 (nt)
B
Size of ds region of masking DNA
a
M
b
1.2 1.0 0.8 0.6 0.4 0.2
5
6
7
9
11
6
7
9
11
13
15
17 (bp)
1.2
Relative Enzyme Activity
A
are provided in the Fig. 1 legend. DNA hybrids formed by hybridization of the KS primer and the complementary DNA strands provide various kinds of partially hybridized DNA molecules with a double-stranded region and a noncomplementary single-stranded region (overhang). These constructs contain a 30 overhang [KS (17-mer)/16-mer hybrid], blunt end [KS (17-mer)/17-mer hybrid], or 50 overhangs with 1- to 51-nt overhangs [KS (17-mer)/18-mer to 65-mer hybrids]. The size of the double-stranded region of the masking DNA molecule was governed by the KS primer used: 17 bp, except for KS (17-mer)/65-mer hybrids, in which it is 16 bp. The largest hybrid in this group, KS (17-mer)/65-mer, contains 50 overhangs at both sides, while other hybrids contain a blunt end and a 50 overhang. These are noted as 1, 0, 1, 2, 3, 5, 7, 9, and 51 50 overhangs in Fig. 1A. Taq DNA polymerase was protected by binding one of these partial DNA hybrids to the active site and immobilized on the Au surface. PCR was performed using the resulting immobilized Taq DNA polymerase and the results are shown in Fig. 1A. We used the activity of PIM Taq polymerase masked with the KS (17-mer)/65-mer hybrid (denoted as 51 in Fig. 1A) as a control for this experiment (100% activity). Relative activity shown in the figure is with respect to the amount of the PCR product obtained from the control. No amplicon was observed when Taq DNA polymerase was masked with a DNA hybrid having a 30 overhang (denoted as 1 in Fig. 1A) or a blunt end (denoted as 0 in Fig. 1A). A relative activity of about 40% was obtained when the KS (17-mer)/20-mer hybrid was used as a masking molecule (denoted as 3 in Fig. 1A). Approximately 67% relative activity was generated stably when the KS (17-mer)/22-mer DNA hybrid was used (denoted as 5 in Fig. 1A). About 84 and 95% relative activity was obtained with KS (17-mer)/24-mer and KS (17-mer)/26-mer hybrids, which contain a 7- and a 9-nt 50 overhang (denoted as 7 or 9 in Fig. 1A), respectively. Quantitative densitometry analysis of DNA amplicons is shown in Fig. 1Ab.
1.0 0.8 0.6 0.4 0.2 0.0
0.0 -1
0
1
2
3
5
7
9
Size of 5' overhang (nucleotide)
51
5
13
15
17
Size of double-stranded DNA region (bp)
Fig.1. Effect of the masking molecule on the active-site masking of Taq DNA polymerase. (A) The masking DNA molecule containing a 30 overhang (1), blunt end (0), or various sizes of 50 overhang (1–51) was generated by hybridization of oligomeric DNA molecules and used for active-site protection of Taq DNA polymerase during the immobilization process. (B) The masking molecules were produced by hybridization of single-stranded DNA molecules. Resulting masking molecules were designed to contain a 9-nt 50 overhang and various sizes of double-stranded region (5–17 bp). The activity of immobilized Taq DNA polymerase was measured by PCR amplification and normalized with respect to the control described in the text. Electrophoresis (a) and quantitative analysis results (b) are shown. Primers used for the experiments shown in (A) were 26-mer (50 -TTCTTTTATCGATACCGTCGACCTCG-30 ), 24-mer (50 -CTTTTATCGATACCGTCGACCTCG-30 ), 22-mer (50 -TTTATCGATACCGTCGACCTCG-30 ), 20-mer (50 -TATCGATACCGTCGACCTCG-30 ), 19-mer (50 -ATCGATACCGTCGACCTCG-30 ), 18-mer (50 -TCGATACCGTCGACCTCG-30 ), 17-mer (50 -CGATACCGTCGACCTCG-30 ), and 16-mer (50 -GATACCGTCGACCTCG-30 ). Primer pairs used for (B) were 15-mer and 24-mer (50 -AGGTCGACGGTATCG-30 and 50 -TTCTTTTATCGATACCGTCGACCT-30 ), 13-mer and 22-mer (50 -GTCGACGGTATCG-30 and 50 -TTCTTTTATCGATACCGTCGAC-30 ), 11-mer and 20-mer (50 -CGACGGTATCG-30 and 50 -TTCTTTTATCGATACCGTCG-30 ), 9-mer and 18-mer (50 -ACGGTATCG-30 and 50 -TTCTTTTATCGATACCGT-30 ), 7-mer and 16-mer (50 -GGTATCG-30 and 50 -TTCTTTTATCGATACC-30 ), 6-mer and 15-mer (50 -GTATCG-30 and 50 -TTCTTTTATCGATAC-30 ), and 5-mer and 14-mer (50 -TATCG-30 and 50 -TTCTTTTATCGATA-30 ).
141
Notes & Tips / Anal. Biochem. 432 (2013) 139–141
Second, the optimal size of the double-stranded region of the masking DNA molecule was determined. The size of a singlestranded DNA was varied from a 5- to a 15-mer and that of the complementary single-stranded DNA was varied from a 14- to a 24-mer. These DNA molecules were hybridized to generate partially double-stranded DNA molecules that had a fixed 9-nt single-stranded 50 overhang and a variable double-stranded region 5 to 17 bp in size. Primer names and sequences are shown in the Fig. 1 legend. Again, the activity of PIM Taq polymerase masked with KS (17-mer)/65-mer hybrids was used as a control. As shown in Fig. 1B, the DNA amplicon was observed as faint bands when the masking DNA molecule had a double-stranded region 5–7 bp in size. A relative activity of about 50% to 90% was observed when the masking DNA molecule had a double-stranded region of 9– 15 bp, compared to the control [KS (17-mer)/65-mer hybrid]. The amount of the DNA amplicon generated was quantitatively analyzed by densitometry and is depicted in Fig. 1Bb. These data suggest that the masking molecule should contain at least about 5–7 nt of the 50 overhang region and at least about 11–13 bp of the double-stranded region to achieve 70% relative activity for the immobilized Taq DNA polymerase. It was shown by crystal structure that Taq DNA polymerase formed a stable complex with a DNA hybrid (having 8 bp of a duplex and 8 nt of a 50 overhang region) bound to the polymerase site [4]. Our results generally agree well with the crystal structure results [4] in terms of the size of the duplex and 50 overhang regions. The slight differences observed for the size of the duplex and 50 overhang regions are probably caused by the differences in the experimental conditions, i.e., the static conditions in the crystal structure experiment vs the dynamic reaction conditions in the immobilization experiment. It is noticeable that the 11- to 13-bp size is just enough to cover one double-helix rotation of a DNA molecule and also it is about half the diameter of the Taq DNA polymerase. These observed size criteria suggest that the active-site protection is mediated by a molecular-level protection of the active site by the masking DNA molecule, leading to preferential formation of a covalent bond away from the active site. Over 1.2 kb of amplicon and RT-PCR products were generated from PIM Taq DNA polymerase One of the critical points of application for the immobilized Taq DNA polymerase would be the accessibility of the immobilized Taq DNA polymerase to a variety of DNA templates. We tried to amplify relatively larger amplicons from larger templates using the immobilized Taq DNA polymerase. The template used for this experiment was a plasmid DNA, pBluescriptII KS(+), and primers that can anneal to specific locations of the plasmid were used to generate PCR amplicons from 68 to 1258 bp. As shown in Fig. 2, the immobilized Taq DNA polymerase was able to generate amplicons of targeted sizes. It is noticeable that the immobilized Taq DNA polymerase can generate an amplicon over 1.2 kb in size. The intensity of DNA amplicon bands was similar to the solution-phase PCR (Fig. 2, left). The immobilized Taq DNA polymerase was also tested for reverse transcription–PCR and it produced amplicons with intensity comparable to that of the solution-phase PCR (data not shown).
Soln 1 2
3
4
PIM 5
6
7 M
1 2
3
4
5
6
7
(bp) ---1,258 --- 803 --- 413 --------- 224 --- 293 --------- 164 --- 68
Fig.2. Amplification range of PIM Taq DNA polymerase. Solid-phase PCR using the PIM Taq DNA polymerase was performed with a template, pBluescriptII KS(+). Primers used were SK and KS for 68 bp, T7 and T3 (50 -AATACGACTCACTATAG-30 and 50 -ATTAACCCTCACTAAAG-30 ) for 164 bp, Universal and Reverse (50 -GTAAAACGACGGCCAGT-30 and 50 -AACAGCTATGACCATG-30 ) for 224 bp, PvuII (50 -TGGCGAAAGGGGGATGT-30 ) and Reverse for 293 bp, NaeI (50 -GGCGAACGTGGCGAGAA-30 ) and KS for 413 bp, SspI (50 -TTTTGTTAAAATTCGCG-30 ) and Reverse for 803 bp, ScaI (50 -ACTCAACCAAGTCATTC-30 ) and Reverse for 1258 bp amplification. Soln, solution-phase PCR; PIM, solid-phase PCR with protected immobilized enzyme; M, molecular weight marker.
In this study, we optimized the masking DNA molecule for active-site protection during the immobilization process of Taq DNA polymerase. The minimum size of the 50 overhang of the masking DNA hybrid was 5–7 nt to have approximately 70% relative activity compared to an immobilized enzyme protected with a large masking molecule. The minimal double-stranded region was 11–13 bp to have approximately 70% relative activity. It was also shown that amplicons over 1.2 kb could be generated by PIM Taq DNA polymerase even though there is considerable spatial limitation of immobilized enzymes. There have been attempts at solid-phase PCR amplification [5,6] and most of these attempts utilized immobilized primers rather than enzymes because of the simplicity of the immobilization method and stability of the DNA molecules during the immobilization process. These methods are limited to amplification of a specific target that is determined by the immobilized primers. However, our data suggest that solidphase PCR can be performed with PIM Taq DNA polymerase and thus a protein chip developed by this method is not limited to predesigned targets but can be applicable to a variety of targets. References [1] G. Lim, H. Hwang, J. Kim, Protected immobilization of Taq DNA polymerase by active site masking on self-assembled monolayers of x-functionalized thiols, Anal. Biochem. 419 (2011) 205–210. [2] D. Li, Q. He, Y. Cui, L. Duan, J. Li, Immobilization of glucose oxidase onto gold nanoparticles with enhanced thermostability, Biochem. Biophys. Res. Commun. 355 (2007) 488–493. [3] H. Wang, X. Wang, X. Zhang, X. Qin, Z. Zhao, Z. Miao, N. Huang, Q. Chen, A novel glucose biosensor based on the immobilization of glucose oxidase onto gold nanoparticles-modified Pb nanowires, Biosens. Bioelectron. 25 (2009) 142–146. [4] S.H. Eom, J. Wang, T.A. Steitz, Structure of Taq polymerase with DNA at the polymerase active site, Nature 382 (1996) 278–281. [5] H. Huang, P. Xiao, Z. Qi, Y. Bu, W. Liu, G. Zhou, A gel-based solid-phase amplification and its application for SNP typing and sequencing on-chip, Analyst 134 (2009) 2434–2440. [6] Y. Sun, R. Dhumpa, D.D. Bang, J. Høgberg, K. Handberg, A. Wolff, A lab-on-a-chip device for rapid identification of avian influenza viral RNA by solid-phase PCR, Lab Chip 11 (2011) 1457–1463.
Contents lists available at SciVerse ScienceDirect
Analytical Biochemistry journal homepage: www.elsevier.com/locate/yabio
Notes & Tips
Optimization of the masking molecule for active-site-protected immobilization of Taq DNA polymerase and its application Gumjung Lim a, Joungmok Kim b, Jeong Hee Kim b,⇑ a b
Department of Chemistry, Kyung Hee University, Seoul 130-701, Republic of Korea Department of Oral Biochemistry and Molecular Biology, Kyung Hee University, Seoul 130-701, Republic of Korea
a r t i c l e
i n f o
Article history: Received 11 July 2012 Received in revised form 19 September 2012 Accepted 24 September 2012 Available online 1 October 2012 Keywords: Active-site protection Taq DNA polymerase Masking molecule Immobilization
a b s t r a c t The method of oriented and activity-preserved immobilization of biologically active proteins based on concepts of active-site masking and kinetic control was further developed in this study. Minimal requirements for the masking DNA molecule were found to be a 50 overhang of 5–7 nucleotides and a doublestranded region of 11–13 bp to retain approximately 70% of the enzyme activity. The amplification range of protected immobilized (PIM) Taq DNA polymerase was over 1.2 kb. These data suggest that PIM Taq DNA polymerase can be used for various commercial applications. Ó 2012 Elsevier Inc. All rights reserved.
We previously reported a new effective immobilization method that involves active-site protection and kinetic control of immobilization density using Taq DNA polymerase as a model system [1]. Taq DNA polymerase is an enzyme whose substrate is much larger than those of other enzymes. The immobilization of such enzymes on a solid surface with preserved enzyme activity has been a challenging subject, while that of enzymes with smaller substrates such as glucose has been more readily achieved [2,3]. In the new immobilization method, the active site of the enzyme is protected with a masking molecule, and the masking molecule preferentially facilitates covalent coupling of the enzyme to the surface at a location away from the active site without relying upon a site-specific immobilization scheme. In this study, the masking DNA molecule for protection of the active site of Taq DNA polymerase during the immobilization process was further optimized to explore application possibilities of the active-site-protected immobilized (PIM) Taq DNA polymerase to the commercial level and the results are reported. Materials and methods Chemicals and reagents The following chemicals were purchased and used for the immobilization reaction: (D,L)-thioctic acid (Aldrich, USA), ⇑ Corresponding author. Fax: +82 2 961 0915. E-mail address: [email protected] (J.H. Kim). 0003-2697/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.ab.2012.09.033
1-ethyl-3-[3-(dimethylamino)propyl]carbamide (Sigma, USA), and N-hydroxysuccinimide (Aldrich). Chemicals used for the selfassembled monolayer (SAM) fabrication, 12-mercaptododecanoic acid [HS(CH2)11COOH] and 6-mercapto-1-hexanol [HS(CH2)6OH], were purchased from Aldrich. Au-coated slides were purchased from EMF Corp. (USA). Taq DNA polymerase and deoxynucleotide mixture were from Applied Biosystems (USA) and Takara (Japan), respectively. Primers were synthesized from Bioneer (Korea). Other chemicals were purchased from Sigma or from other common sources. Immobilization of active-site-protected Taq DNA polymerase and solid-phase polymerase chain reaction (PCR) The Au substrate used was a glass plate 3.0 5.0 mm in size on which Au was vacuum-deposited to about 1000 Å thickness. The fabrication of SAM on the Au surface and immobilization of Taq DNA polymerase were performed as reported previously [1], except that the masking DNA molecule was changed to DNA molecules with various lengths and structures as described below. PCR was performed as described in Lim et al. [1]. A 65-base single-stranded DNA (65-mer, single-stranded DNA; 30 -CCAGCTGCCATAGCTATTTTCTTTTCTTTCTTAAGTTCTTTTCTTTTCCTAGGTGATCAAGATCT-50 , 25 fmol) was used as a template, and KS and SK primers (50 -CGAGGTCGACGGTATCG-30 and 50 -TCTAGAACTAGTGGATC-30 , 10 pmol each) were used for PCR in a reaction volume of 50 ll. The temperature cycle was set as follows: hot start step, 94 °C, 10 min; 35 cycles of 94 °C, 30 s, 50 °C, 60 s, 72 °C, 30 s, unless
140
Notes & Tips / Anal. Biochem. 432 (2013) 139–141
specified otherwise. For masking molecule optimization experiments, 35 cycles of PCR were performed: 94 °C, 30 s, 42 °C, 30 s, 72 °C, 30 s. For larger amplicons, pBluescriptII KS(+) was used as a template and primer sets used were as specified in the figure legend. PCR cycling conditions were as follows: hot start step, 94 °C, 10 min; 35 cycles of 94 °C, 30 s, 50 °C, 30 s, 72 °C, 2 min. Final extension was performed at 72 °C for 10 min. A part of the PCR solution was sampled after the PCR and analyzed by agarose gel electrophoresis. The PCR products were visualized by fluorescence from ethidium bromide staining and quantified with a densitometer (Ultra-Lum Imaging system, USA). All experiments were performed at least in triplicate. Results and discussion Optimization of masking molecule for active-site protection of Taq DNA polymerase To prepare an immobilization matrix, a SAM of x-functionalized thiols on a Au surface was formed as described previously [1]. The active site of the target enzyme, Taq DNA polymerase, was protected by masking with a partially double-stranded DNA molecule and then immobilized through amide bonds to the activated SAM. The amount of the Taq DNA polymerase used (0.75 pmol) corresponded to the amount that can form three monolayers on the area of 3 5 mm of the Au substrate. First, the minimum required size of the noncomplementary single-stranded region of the masking DNA molecule for protecting the active site of Taq DNA polymerase was determined. The size of a single-stranded DNA was fixed as a 17-mer by using the KS primer, and the size of the complementary single-stranded DNA was varied from a 16- to a 65-mer. Sequences of these DNA molecules Size of 5’ overhang of masking DNA
a
Relative Enzyme Activity
b
M -1
0 1
2 3
5
7
9
51 (nt)
B
Size of ds region of masking DNA
a
M
b
1.2 1.0 0.8 0.6 0.4 0.2
5
6
7
9
11
6
7
9
11
13
15
17 (bp)
1.2
Relative Enzyme Activity
A
are provided in the Fig. 1 legend. DNA hybrids formed by hybridization of the KS primer and the complementary DNA strands provide various kinds of partially hybridized DNA molecules with a double-stranded region and a noncomplementary single-stranded region (overhang). These constructs contain a 30 overhang [KS (17-mer)/16-mer hybrid], blunt end [KS (17-mer)/17-mer hybrid], or 50 overhangs with 1- to 51-nt overhangs [KS (17-mer)/18-mer to 65-mer hybrids]. The size of the double-stranded region of the masking DNA molecule was governed by the KS primer used: 17 bp, except for KS (17-mer)/65-mer hybrids, in which it is 16 bp. The largest hybrid in this group, KS (17-mer)/65-mer, contains 50 overhangs at both sides, while other hybrids contain a blunt end and a 50 overhang. These are noted as 1, 0, 1, 2, 3, 5, 7, 9, and 51 50 overhangs in Fig. 1A. Taq DNA polymerase was protected by binding one of these partial DNA hybrids to the active site and immobilized on the Au surface. PCR was performed using the resulting immobilized Taq DNA polymerase and the results are shown in Fig. 1A. We used the activity of PIM Taq polymerase masked with the KS (17-mer)/65-mer hybrid (denoted as 51 in Fig. 1A) as a control for this experiment (100% activity). Relative activity shown in the figure is with respect to the amount of the PCR product obtained from the control. No amplicon was observed when Taq DNA polymerase was masked with a DNA hybrid having a 30 overhang (denoted as 1 in Fig. 1A) or a blunt end (denoted as 0 in Fig. 1A). A relative activity of about 40% was obtained when the KS (17-mer)/20-mer hybrid was used as a masking molecule (denoted as 3 in Fig. 1A). Approximately 67% relative activity was generated stably when the KS (17-mer)/22-mer DNA hybrid was used (denoted as 5 in Fig. 1A). About 84 and 95% relative activity was obtained with KS (17-mer)/24-mer and KS (17-mer)/26-mer hybrids, which contain a 7- and a 9-nt 50 overhang (denoted as 7 or 9 in Fig. 1A), respectively. Quantitative densitometry analysis of DNA amplicons is shown in Fig. 1Ab.
1.0 0.8 0.6 0.4 0.2 0.0
0.0 -1
0
1
2
3
5
7
9
Size of 5' overhang (nucleotide)
51
5
13
15
17
Size of double-stranded DNA region (bp)
Fig.1. Effect of the masking molecule on the active-site masking of Taq DNA polymerase. (A) The masking DNA molecule containing a 30 overhang (1), blunt end (0), or various sizes of 50 overhang (1–51) was generated by hybridization of oligomeric DNA molecules and used for active-site protection of Taq DNA polymerase during the immobilization process. (B) The masking molecules were produced by hybridization of single-stranded DNA molecules. Resulting masking molecules were designed to contain a 9-nt 50 overhang and various sizes of double-stranded region (5–17 bp). The activity of immobilized Taq DNA polymerase was measured by PCR amplification and normalized with respect to the control described in the text. Electrophoresis (a) and quantitative analysis results (b) are shown. Primers used for the experiments shown in (A) were 26-mer (50 -TTCTTTTATCGATACCGTCGACCTCG-30 ), 24-mer (50 -CTTTTATCGATACCGTCGACCTCG-30 ), 22-mer (50 -TTTATCGATACCGTCGACCTCG-30 ), 20-mer (50 -TATCGATACCGTCGACCTCG-30 ), 19-mer (50 -ATCGATACCGTCGACCTCG-30 ), 18-mer (50 -TCGATACCGTCGACCTCG-30 ), 17-mer (50 -CGATACCGTCGACCTCG-30 ), and 16-mer (50 -GATACCGTCGACCTCG-30 ). Primer pairs used for (B) were 15-mer and 24-mer (50 -AGGTCGACGGTATCG-30 and 50 -TTCTTTTATCGATACCGTCGACCT-30 ), 13-mer and 22-mer (50 -GTCGACGGTATCG-30 and 50 -TTCTTTTATCGATACCGTCGAC-30 ), 11-mer and 20-mer (50 -CGACGGTATCG-30 and 50 -TTCTTTTATCGATACCGTCG-30 ), 9-mer and 18-mer (50 -ACGGTATCG-30 and 50 -TTCTTTTATCGATACCGT-30 ), 7-mer and 16-mer (50 -GGTATCG-30 and 50 -TTCTTTTATCGATACC-30 ), 6-mer and 15-mer (50 -GTATCG-30 and 50 -TTCTTTTATCGATAC-30 ), and 5-mer and 14-mer (50 -TATCG-30 and 50 -TTCTTTTATCGATA-30 ).
141
Notes & Tips / Anal. Biochem. 432 (2013) 139–141
Second, the optimal size of the double-stranded region of the masking DNA molecule was determined. The size of a singlestranded DNA was varied from a 5- to a 15-mer and that of the complementary single-stranded DNA was varied from a 14- to a 24-mer. These DNA molecules were hybridized to generate partially double-stranded DNA molecules that had a fixed 9-nt single-stranded 50 overhang and a variable double-stranded region 5 to 17 bp in size. Primer names and sequences are shown in the Fig. 1 legend. Again, the activity of PIM Taq polymerase masked with KS (17-mer)/65-mer hybrids was used as a control. As shown in Fig. 1B, the DNA amplicon was observed as faint bands when the masking DNA molecule had a double-stranded region 5–7 bp in size. A relative activity of about 50% to 90% was observed when the masking DNA molecule had a double-stranded region of 9– 15 bp, compared to the control [KS (17-mer)/65-mer hybrid]. The amount of the DNA amplicon generated was quantitatively analyzed by densitometry and is depicted in Fig. 1Bb. These data suggest that the masking molecule should contain at least about 5–7 nt of the 50 overhang region and at least about 11–13 bp of the double-stranded region to achieve 70% relative activity for the immobilized Taq DNA polymerase. It was shown by crystal structure that Taq DNA polymerase formed a stable complex with a DNA hybrid (having 8 bp of a duplex and 8 nt of a 50 overhang region) bound to the polymerase site [4]. Our results generally agree well with the crystal structure results [4] in terms of the size of the duplex and 50 overhang regions. The slight differences observed for the size of the duplex and 50 overhang regions are probably caused by the differences in the experimental conditions, i.e., the static conditions in the crystal structure experiment vs the dynamic reaction conditions in the immobilization experiment. It is noticeable that the 11- to 13-bp size is just enough to cover one double-helix rotation of a DNA molecule and also it is about half the diameter of the Taq DNA polymerase. These observed size criteria suggest that the active-site protection is mediated by a molecular-level protection of the active site by the masking DNA molecule, leading to preferential formation of a covalent bond away from the active site. Over 1.2 kb of amplicon and RT-PCR products were generated from PIM Taq DNA polymerase One of the critical points of application for the immobilized Taq DNA polymerase would be the accessibility of the immobilized Taq DNA polymerase to a variety of DNA templates. We tried to amplify relatively larger amplicons from larger templates using the immobilized Taq DNA polymerase. The template used for this experiment was a plasmid DNA, pBluescriptII KS(+), and primers that can anneal to specific locations of the plasmid were used to generate PCR amplicons from 68 to 1258 bp. As shown in Fig. 2, the immobilized Taq DNA polymerase was able to generate amplicons of targeted sizes. It is noticeable that the immobilized Taq DNA polymerase can generate an amplicon over 1.2 kb in size. The intensity of DNA amplicon bands was similar to the solution-phase PCR (Fig. 2, left). The immobilized Taq DNA polymerase was also tested for reverse transcription–PCR and it produced amplicons with intensity comparable to that of the solution-phase PCR (data not shown).
Soln 1 2
3
4
PIM 5
6
7 M
1 2
3
4
5
6
7
(bp) ---1,258 --- 803 --- 413 --------- 224 --- 293 --------- 164 --- 68
Fig.2. Amplification range of PIM Taq DNA polymerase. Solid-phase PCR using the PIM Taq DNA polymerase was performed with a template, pBluescriptII KS(+). Primers used were SK and KS for 68 bp, T7 and T3 (50 -AATACGACTCACTATAG-30 and 50 -ATTAACCCTCACTAAAG-30 ) for 164 bp, Universal and Reverse (50 -GTAAAACGACGGCCAGT-30 and 50 -AACAGCTATGACCATG-30 ) for 224 bp, PvuII (50 -TGGCGAAAGGGGGATGT-30 ) and Reverse for 293 bp, NaeI (50 -GGCGAACGTGGCGAGAA-30 ) and KS for 413 bp, SspI (50 -TTTTGTTAAAATTCGCG-30 ) and Reverse for 803 bp, ScaI (50 -ACTCAACCAAGTCATTC-30 ) and Reverse for 1258 bp amplification. Soln, solution-phase PCR; PIM, solid-phase PCR with protected immobilized enzyme; M, molecular weight marker.
In this study, we optimized the masking DNA molecule for active-site protection during the immobilization process of Taq DNA polymerase. The minimum size of the 50 overhang of the masking DNA hybrid was 5–7 nt to have approximately 70% relative activity compared to an immobilized enzyme protected with a large masking molecule. The minimal double-stranded region was 11–13 bp to have approximately 70% relative activity. It was also shown that amplicons over 1.2 kb could be generated by PIM Taq DNA polymerase even though there is considerable spatial limitation of immobilized enzymes. There have been attempts at solid-phase PCR amplification [5,6] and most of these attempts utilized immobilized primers rather than enzymes because of the simplicity of the immobilization method and stability of the DNA molecules during the immobilization process. These methods are limited to amplification of a specific target that is determined by the immobilized primers. However, our data suggest that solidphase PCR can be performed with PIM Taq DNA polymerase and thus a protein chip developed by this method is not limited to predesigned targets but can be applicable to a variety of targets. References [1] G. Lim, H. Hwang, J. Kim, Protected immobilization of Taq DNA polymerase by active site masking on self-assembled monolayers of x-functionalized thiols, Anal. Biochem. 419 (2011) 205–210. [2] D. Li, Q. He, Y. Cui, L. Duan, J. Li, Immobilization of glucose oxidase onto gold nanoparticles with enhanced thermostability, Biochem. Biophys. Res. Commun. 355 (2007) 488–493. [3] H. Wang, X. Wang, X. Zhang, X. Qin, Z. Zhao, Z. Miao, N. Huang, Q. Chen, A novel glucose biosensor based on the immobilization of glucose oxidase onto gold nanoparticles-modified Pb nanowires, Biosens. Bioelectron. 25 (2009) 142–146. [4] S.H. Eom, J. Wang, T.A. Steitz, Structure of Taq polymerase with DNA at the polymerase active site, Nature 382 (1996) 278–281. [5] H. Huang, P. Xiao, Z. Qi, Y. Bu, W. Liu, G. Zhou, A gel-based solid-phase amplification and its application for SNP typing and sequencing on-chip, Analyst 134 (2009) 2434–2440. [6] Y. Sun, R. Dhumpa, D.D. Bang, J. Høgberg, K. Handberg, A. Wolff, A lab-on-a-chip device for rapid identification of avian influenza viral RNA by solid-phase PCR, Lab Chip 11 (2011) 1457–1463.