3.2 Material and methods
3.3.3 The fluorescently modified λ - DNA and the sample purity 44
In order to find a complementary test method for the dot-blot technique we tested also microarray reader to estimate the free oligo content of our λ - DNA samples. This was done by modifying theλ- DNA molecules with oligos carrying fluorophore molecule (Cy3) and measuring with microarray reader the emitted fluorescence. Each, with fluorophore end-modified, sample was compared with a sample containing equimolarλ- DNA molecules and oligo molecules in same mass concentration as end-modified sample. By comparing the measured intensities we were able to estimate the filtering efficiency. The microarray scans were analyzed by summing up the whole intensity that each sample spot emitted (see Fig. 3.2 (b)).
First we redid the same experiment14 as was already done with dot-blot: we compared the fluorescence emitted by equimolar mixture of λ - DNA and oligo with λ - DNA sample purified over NICKT M column. The eluation solution with NICKT M column was this time TBE. In this experiment TBE was chosen as a buffer since it does not contain NaCl and therefore there should be no measurement distorting NaCl crystals forming as the λ - DNA – oligo solution dries on the glass slide. Here we got as a result (data not shown) that the signal was on average 65 times higher from the sample purified with NICKT M column than from the equimolar solution.
In order to further purify the samples from the oligos, we used PEG pre-cipitation protocol15 after the NICKT M column filtering. In this method the λ -DNA–oligo solution is mixed with PEG which causes the DNA to precipitate and leaves oligos and salts in the supernatant. In the first step PEG and NaCl were added to the DNA–oligo solution so that the end concentration was 10 % of PEG and 0.5 M NaCl. This solution was mixed gently with pipette and then put for 30 minutes on ice. After that the solution was centrifuged for 15 minutes at room temperature with maximum speed (15000×g). The centrifugation caused DNA to concentrate at the bottom of the vial. The concentrated DNA was washed with 70 % and 96 % ethanol, respectively and dried with SpeedVac16 or just by letting ethanol to evaporate at room temperature. In the final step DNA was eluted with 100 µl of TBE and diluted to desired measurement concentrations.
Additionally the samples were sonicated before dilutions in order to cut the DNA strands into small pieces. This is done in order to distribute DNA over the whole vial homogeneously so that the concentration determination is more accurate.
In the experiments we used PEG 8000 (average molecular weight 8000 g/mol) and PEG 3350 (average molecular weight 3350 g/mol) for precipitation. The
14The DNA concentrations were 43 ng/µl, 21.5 ng/µl, 10.75 ng/µl and only TBE buffer.
15Mikrobiologisches Praktikum f¨ur Fortgeschrittene I, Universit¨at Erlangen (see Appendix B).
16The SpeedVac is used to concentrate samples by letting the solvent to evaporate in vacuum.
Figure 3.6: Cy3-modified DNA was purified by precipitating it with PEG 3350 or with PEG 8000. Additionally the samples were also filtered with NICKT M column. The emitted fluorescence after filtering was measured with microarray reader and compared with signal from equimolar λ - DNA–oligo mixture at the same mass concentration. When DNA was purified with PEG3350the signal was on average4.4times higher than the signal of equimolar DNA-oligo mixture and when the same experiment was done with PEG 8000 the signal was on average 1.3 times higher than the signal of equimolar DNA-oligo mixture.
results are presented in Fig. 3.6 where it is seen that with PEG 8000 purified DNA solution gives slightly, on average 1.3 times, higher signal than the equimolar DNA–oligo mixture. When the sample was purified with PEG 3350 the measured signal was on average 4.4 times higher than the equimolar DNA–oligo mixture.
This result shows that the filtering of the samples through PEG 8000 precipitation removes oligos very effectively since after precipitation there is only ∼ 1.3 oligo molecules present per singleλ - DNA molecule.
Furthermore we tested also the functionality of end-modified DNA after pre-cipitation with PEG. This was done so that we precipitated and filtered with NICKT M column biotin-modified DNA and later incubated it on streptavidin sur-face. Here we noticed that when the precipitation was done before the NICKT M column filtering we got normally dense carpets (see for example Fig. 4.2 (b)).
When the NICKT M column filtering was done before the PEG precipitation, the carpet densities were dilute (see for example Fig. 4.2 (d)). Additionally we tested the PEG precipitated DNA also with the stretching setup and we did not notice any difference to non-precipitated DNA samples. As an open question, however, stays whether the precipitation procedure causes DNA strand breaks.
In conclusion the results show that through precipitation we managed to get
almost oligo free DNA samples. The filtering effect of PEG 3350 is not as good as with PEG 8000. Furthermore it seems that the additional filtering with NICKT M column should be done after precipitation to ensure full removal of PEG and thereby full functionality of end-modified DNA.
3.3.4 High quality PCR DNA for force measurements
The advantages of the use of PCR in sample preparation, in comparison to mod-ifyingλ - DNA with oligos, are that the product has only user defined length (no concatemers) and the primers are covalently joined to the backbone. Addition-ally with PCR it is possible to produce short fragments of DNA which carry high amount of modifications as modified bases can be used. These fragments can then be ligated for example with λ - DNA molecule and so they provide a possibility to constrain the rotation of the λ - DNA molecules upon stretching (see L´eger et al. [41]). Additionally for the evanescent wave experiments presented in chapter 6, we needed short single end-modified DNA molecules and the easiest way to produce them was to use PCR.
The limitation to use PCR is the fact that length of the PCR products has been limited under ∼ 5 kbp and this is too small for example for the birefrin-gence experiment described in the general introduction 1. However, recent de-velopments in PCR techniques [42] [43] have made it possible to manufacture PCR products up to the length of λ - DNA. Our work with PCR is still very much underway since finding the optimal conditions for DNA production with PCR can be difficult. Up to now we have been able to produce 2 kbp DNA with one end modification and 20 kbp DNA with biotin- and thiol-modifications. The results are shown in Fig. 3.7 where in (a) the 2 kbp product is visible on the right side of a marker which shows that the PCR product is obtained correctly. Fig.
3.7 (b) shows the biotin- and thiol-modified 20 kbp long PCR product running on the lane (iii). Here we have used the same marker as in (a) on lane (i) which again confirms that the PCR has functioned correctly. Further confirmation is gained from the lane (ii) on which unmodified 20 kbp PCR produced DNA is running. The unmodified 20 kbp DNA was produced in order to optimize the PCR parameters and so to save modified primer material.
The biotin- and thiol-modified 20 kbp long DNA was also tested on corre-sponding surfaces for its functionality as a result we achieved similar carpets as with λ - DNA. The functionality will be further tested in stretching experiments as presented in chapter 2.
In conclusion we have established a working protocol for relatively long end-modified PCR produced DNA (20 kbp). Furthermore all the protocols developed to purify the λ - DNA samples can also be adapted for the PCR products so we have broad machinery for future experiment where for example the rotation of the DNA molecules can be constrained.
21226 bp
2027 bp
1904 bp
Template Bluescript 2KS
PCR product:
2 kbp Error product from 1st PCR cycle Marker PCR product
Wells
3530 bp
(a)
Marker:
21226 bp
PCR product:
biotin- and thiol-modified, 20 kbp Wells
(i) (ii) (iii)
(b)
Figure 3.7: a) Gel electrophoresis was used to study PCR products. The cor-rectness of2 kbp long PCR product DNA was estimated with a marker running on a lane left to the product. The2kbp long product, template (Bluescript2KS, 2961 bp) and an error product apparently originating from the first PCR cycle are highlighted with red lines. b) For the biotin- and thiol-modified 20 kbp long DNA the same analysis was also done as in (a). The marker on the lane (i) con-firms that the20kbp PCR product has the correct length. Further confirmation is gained from the lane (ii) where unmodified 20 kbp long PCR produced DNA is found. This DNA was produced in order to find the right parameters for the PCR protocol. Finally on the lane (iii) is the biotin- and thiol-modified 20kbp long DNA.
3.4 Conclusions
In this chapter we have dwelled into the problems of sample preparation for DNA stretching experiments. By using fluorescent Cy3 oligos and microarray reader, we found that our DNA sample preparation was not optimized since after filtering with NICKT M column there was still ∼ 65 oligos per modifiedλ - DNA present in the stock solution. The problem of the free oligos was solved by using PEG precipitation in addition to filtering. After the renewed filtering we found only 1.3 oligos perλ- DNA molecule. Furthermore we did not observe the precipitation to harm the functionality of DNA or its end-modification. Furthermore by using field inversion gel electrophoresis (FIGE) we confirmed the observation from chapter 2 that ourλ- DNA samples contain concatemers. The amount of concatemers was found to be in the range of few percent. This result is in good agreement with the estimate which was achieved from the rupture length distributions. Finally we have shown that PCR could be used for sample preparation of long end-modified DNA. The use of PCR would circumvent the problem of oligo ligation to the DNA molecule as here the whole DNA is synthesized. PCR techniques open also new possibilities to modify DNA molecules with several end-modifications. The work with long PCR products is, however, subject for further investigations and optimization.
Covalent end-attachment of end-modified DNA on a solid substrate
Abstract
In this chapter we will study various end-grafting chemistries to specifically bind λ - DNA onto functionalized surfaces. Further-more we will also study few possibilities to introduce target groups for cross-linkers on surfaces through silanization. Confocal fluores-cence microscopy was used to estimate the grafting efficiency, and the quality of the end-grafts was tested in most of the cases with the setup presented in chapter 2. As a result we provide several working protocols to end-graft λ- DNA on a solid support. Our re-sults are significant for the realization of efficient double-sided end-attachment of DNA for the future force-extension measurements as discussed in general introduction 1.
49
4.1 Introduction
Covalent attachment of DNA molecules onto a solid support has become an in-creasingly important biological tool in recent years. DNA microarray techniques used in drug development and medical diagnostics use typically single-stranded DNA attached at the surface to fish out the complementary DNA strands [44].
Furthermore, as already mentioned in chapter 2, all the single molecule force ex-periments need a stable/covalent bond of the DNA to the surface. There are a lot of similarities in surface chemistries between single molecule force experiments and DNA microarray techniques, namely in most of the cases the hydroxyl groups at the surface of glass or silicon wafer are silanized and then the rest of the chem-istry is built on top of that. What the remaining chemchem-istry then is, depends on the needs of the experiment. In summary a great variety of chemical tools have been developed to end-graft DNA or specifically attach other bio-molecule onto a surface but most of the work is done either with short oligos or with proteins.
Our goal in this study is to find out the techniques which are the most suitable for attaching λ - DNA specifically on a solid support of our choice. When working with a gigantic macromolecule such as λ - DNA, there are some hard criteria for a suitable surface chemistry: (i) the reactive groups have to stay active for a long time and not hydrolyze or inactivate otherwise since the binding kinetics are very slow for λ - DNA (compare chapter 5), (ii) the reactivity of the surface should be low against unfunctionalized DNA so that DNA is not sticking onto a surface, (iii) the reactive groups should be reactive only with one end-modification of the DNA molecule at a time and (iv) finally the reaction chemicals have to be user friendly so that they are not toxic, cheap and as easy as possible to handle.
In the chapter 2, we found that λ - DNA cross-linked over glutaraldehyde and biotin-streptavidin complex to the APTES silanized surface works for the single molecules but to parallelize the stretching for thousands of molecules does not work. This was the case at least for single biotin end-modification. The origin of the problems was found to be: (i) The possibility of physisorption of streptavidin without being chemically linked to the surface since the homobifunc-tional cross-linker glutaraldehyde can react already alone at the surface. (ii) The biotin-streptavidin bond is not stable enough at low loading rates [29] so that the force-plateau could be reached. In this chapter we will present our study of various kinds of possibilities to end-attach λ - DNA on glass. We will start by presenting the various surface chemistry possibilities that we have tried going them through first schematically and then presenting experimental protocols. Fi-nally the surface chemistries, which we found to work with λ - DNA, were tested by stretching single molecules with the setup presented in chapter 2, so that we can be sure that the surface chemistry in question also works as expected.