使用单分子DNA拉伸含量直接观察DNA复制的酶
Summary
我们描述了一个观察单个DNA分子介导的蛋白质的噬菌体复制系统的实时复制的方法。
Abstract
我们描述了一个观察单个DNA分子介导的蛋白质的噬菌体复制系统的实时复制的方法。线性λDNA被修改，有一个链上的生物素，地高辛基团上的同一条电缆的另一端。生物素标记的一端连接到一个官能的玻璃盖玻片和digoxigeninated结束的小珠。一个流细胞表面上这些DNA珠系绳大会允许应用层流珠施加的阻力。因此，被拉伸的DNA靠近和平行的流量（图1）确定力在盖玻片表面。测量长度的DNA监测珠的位置。单双链DNA之间的长度差异，利用获得的实时信息，在交岔路口的复制蛋白的活性。测量珠的位置可以精确测定平仓DNA和聚合（图2）的速率和processivities。
Protocol
1。 DNA复制模板 DNA的反应是一个线性的λDNA寡核苷酸退火形成一个复制叉的修改。此外，生物素连接的λDNA链的结束，一个地高辛基团连接到同一条 1的另一端。 材料：λ噬菌体的DNA，寡核苷酸：生物素化的叉臂（答：5' -生物素- AAAAAAAAAAAAAAAAGAGTACTGTACGATCTAGCATCAATCACAGGGTCAGGTTCGTTATTGTCCAACTTGCTGTCC – 3'），λ互补叉臂（B：5' – GGGCGGCGACCTGGA…
Discussion
重要的是要确保对珠层流曳力不影响酶活性的复制蛋白。举例来说，一个3 PN的力量，对应到0.012毫升/分钟的流速不影响复制的DNA领先链。但它的影响，采取协调DNA合成过程中酶的活性，因此将减少到1.5 PN。拖曳力可以很容易地控制，通过改变流速或流道 5的宽度。
DNA拉伸实验采用双和单链DNA之间的长度差异。在实验中所描述这里的单链DNA双链DNA转换作为一个领先的?…
Acknowledgements
拉伸实验的DNA的发展援助，钟奉李保布莱尼和萨米尔哈姆丹。这项工作是由美国国立卫生赠款查尔斯理查德森（GM54397），安托万车Oijen（GM077248）研究院的支持。
Materials
| Material Name | Tipo | Company | Catalogue Number | Comment |
|---|---|---|---|---|
| Bacteriophage λ DNA | New England Biolabs | N3011L | ||
| DNA Oligonucleotides | Integrated DNA Technologies | |||
| T4 DNA Ligase | New England Biolabs | M0202L | ||
| T4 Polynucleotide Kinase | New England Biolabs | M0201L | ||
| α-digoxigenin Fab | Roche | 11214667001 | ||
| Tosyl Activated Beads | Dynal/Invitrogen | 142-03 | ||
| Magnetic Separator | Invitrogen | Dynal MPC | ||
| 3-aminopropyl-triethoxysilane | Sigma | A3648 | Other aminosilanes can be used or mixed with non-amine reactive silanes for sparser surfaces | |
| Succinimidyl propionate PEG | Nektar | Similar PEGs can be purchased from Nanocs, CreativePEGWorks, etc. | ||
| Biotin-PEG-NHS | Nektar | Similar PEGs can be purchased from Nanocs, CreativePEGWorks, etc. | ||
| Double-sided tape | Grace BioLabs | SA-S-1L | 100 μm thickness | |
| Quartz slide | Technical Glass | 20 mm (W)x 50 mm (L)x 1mm (H) | Size to fit on coverslips. Drill holes with diamond-tip drill bits (DiamondBurs.net) | |
| Polyethylene tubing | Becton Dickinson | 427416 | 0.76 mm ID, 1.22 OD Other size tubing can be substituted. |
|
| Streptavidin | Sigma | S4762 | Make 1 mg/mL solution, 25 μL aliquots in PBS pH 7.3 | |
| Deoxyribonucleotide triphosphate solution mix | New England Biolabs | N0447 | ||
| Inverted Optical Microscope with 10X Objective | Olympus | Olympus IX-51 | ||
| Permament rare-earth magnet | National Imports | www.rare-earth-magnets.com | ||
| CCD Camera | QImaging | Rolera-XR Fast 139 | ||
| Syringe Pump | Harvard Apparatus | 11 Plus | Operate in refill mode to facilitate solution changes | |
| Fiber Illuminator | Thorlabs Inc. | OSL1 |
Referências
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- Sofia, S. J., Premnath, V. V., Merrill, E. W. Poly(ethylene oxide) Grafted to Silicon Surfaces: Grafting Density and Protein Adsorption. Macromolecules. 31, 5059-5070 (1998).
- Tanner, N. A., Loparo, J. J., van Oijen, A. M. Visualizing single-molecule DNA replication with fluorescence microscopy. J Vis Exp. , (2009).
- Hamdan, S. M., Loparo, J. J., Takahashi, M., Richardson, C. C., van Oijen, A. M. Dynamics of DNA replication loops reveal temporal control of lagging-strand synthesis. Nature. 457, 336-339 (2009).
- van Oijen, A. M. Single-molecule kinetics of lambda exonuclease reveal base dependence and dynamic disorder. Science. 301, 1235-1238 (2003).
Tags
EnzymesReplicating DNASingle-molecule DNA Stretching AssayBacteriophage Replication SystemBiotinDigoxigeninFunctionalized Glass CoverslipSmall BeadDNA-bead TethersFlow CellLaminar FlowDrag ForceStretched DNACoverslip SurfaceFlow RateLength MeasurementSingle-stranded DNADouble-stranded DNAReplication ProteinsForkUnwindingPolymerization
Citar este artigo
Kulczyk, A. W., Tanner, N. A., Loparo, J. J., Richardson, C. C., van Oijen, A. M. Direct Observation of Enzymes Replicating DNA Using a Single-molecule DNA Stretching Assay. J. Vis. Exp. (37), e1689, doi:10.3791/1689 (2010).