RM's+Intel+Entry

Here is the version of my report that I sent into Intel (text only, without captions beneath the images, as well): **Rationale and Purpose of Research ** The interest in DNA research has escalated in recent years due to multiple unique properties of DNA. High specificity in the self-assembly of DNA, rigidity, and the ability to create arbitrary sequences of DNA has allowed researches to consider it as a scaffold or for the creation of new technological devices and circuit boards.1-3 These devices could have new properties not present in current silicon based devices such as a combined3 “easy-to-read test for the presence of a genetic sequence” and cantilevers that can scan biological samples for a genetic sequence.4 However, in order to be used as scaffolding, DNA rafts must be formed in predictable ways. If DNA raft chains form according to certain patterns, then what are these patterns and what rules are followed for their formation? In an effort to answer these questions, we constructed DNA raft chains under various physiochemical conditions and analyzed the raft formed. We assessed the conditions that were most conducive to DNA raft formation. Based on the data, previous models used to predict the numbers of DNA rafts in a chain were tested and new models may be proposed to explain the patterns observed. This advances the effort to create DNA platforms that may be used for potential technological devices and circuit boards.  **Pertinent Scientific Literature **  Several research groups have been active in this field. One publication examined the effects of DNA oligonucleotides assembling into rafts and binding on silicon wafers with trenches. APTES (3-aminopropyltriethoxysilane), a substance that binds the DNA to the silicon surface, was deposited in these trenches. The DNA did, indeed, bind to the “stripes” of APTES, resulting in a patterning of the silicon surface,1 while other investigators have used theoretical computer modeling approaches to help develop nanostructures. They have shown that DNA can be “patterned’ in order to have other specifically designated materials bind to it, and have created a computer program from which DNA platforms can be mass-produced.5 Another interesting approach was the use of 2-D structures, created by folding single stranded DNA. This “origami” of DNA can then be combined with other techniques, such as self-assembly, to create more complex structures that can have additional, selected components programmed to bind to it.6 Mathematical literature on condensation polymerization is crucial to this area of research. Algorithmic self-assembly has been explored recently; however, limitations include errors in growth processes and control of variables.7 More specifically, Wallace Carothers derived an equation for step-polymerization that related the degree of conversion (or the percent of the monomers that formed chains) to the number average degree of polymerization (or the number of particles/rafts in the chain).8 Additionally, the Polydispersity Index (PDI), explains the spread of molecular mass in a sample containing polymers.8 Together, these two equations form the basis of much of the analysis conducted in this report.

**Prior Works and Contributions of Others on This Project **  Within the laboratory, research had been conducted to induce DNA plasmids to bind to mica and silicon using APTES. Later on, DNA rafts were substituted for the plasmids, yielding results that demonstrated that the rafts bound to non-APTES areas of a silicon chip 20 times less than the APTES stripes.1 Electron-beam lithography (EBL) and the use of cationic surface-active monomers (SAMs) have been shown to provide effective ways to create scaffolding for nanostructures.1 Other monolayers, such as polyethylene glycol (PEG) and octadecyltrichlorosilane (OTS) have also been employed to bind DNA to silicon, in order to loosen the DNA for the purpose of arranging it. In order to induce conductivity to the DNA, the deposition of gold particles to the DNA scaffolding is being explored. Top-down methods to pattern a surface, and in particular, EBL, are being used to explore methods of patterning surfaces. In short, this is an interdisciplinary field, involving the use of the Atomic Force Microscope (AFM), Scanning Tunneling Microscope (STM), EBL, spectroscopy, computer modeling and quantitative analysis. When I started researching in the lab, the DNA raft had been designed and its sequence had been defined. My objective was to a) identify the ideal physical chemical conditions to produce the DNA rafts; b) discover the mode of polymerization of a specific type of DNA rafts and c) determine if the pattern observed followed some commonly accepted models for macromolecule hybridization. **Methodology **  In order to conduct the research, I had full access to a lab and the guidance of mentors. Therefore, I was fortunate to be able to obtain and use a lab and any necessary materials for my project, which included DNA strands, buffer solutions, nitrogen gas, mica chips, desiccators, and an AFM and its software, which was the Nanoscope IIIa Multimode instrument.  In order to create my samples, the ds (double-stranded) DNA had to be ordered. Figure 1, below, shows the structure of the raft. A Tile <span style="font-size: 12pt; font-family: 'Times New Roman','serif';">4RedA 5’ ACT GGT GGA AGGTTT AAG GTG AAA CTC GAC CTC TAT TCC CTG GCG ATG 3’ <span style="font-size: 12pt; font-family: 'Times New Roman','serif';">4YellowA 5’ CAT CGC CAC CAC CAG TGA GAT 3’ <span style="font-size: 12pt; font-family: 'Times New Roman','serif';">4BlueA 5’ CAG AGG TCT TTC ACC TTA AAC CTT GGG AAT AGA GGT CGA GAC AAG TCG 3’ <span style="font-size: 12pt; font-family: 'Times New Roman','serif';">4GreenA 5’ CGA CTT GTG ACC TCT GTC TTG 3’ <span style="font-size: 12pt; font-family: 'Times New Roman','serif';"> <span style="font-size: 12pt; font-family: 'Times New Roman','serif';">B’Tile <span style="font-size: 12pt; font-family: 'Times New Roman','serif';">4RedB 5’ AGT GAG GAC AGG CAA CGA AGC ATC TC 3’ <span style="font-size: 12pt; font-family: 'Times New Roman','serif';">4YellowB 5’ GCT TCG TTC CCA CGA ACC TAC AGC AAG CCA CGA TAG CTC AGC CTG TCC 3’ <span style="font-size: 12pt; font-family: 'Times New Roman','serif';">4BlueB 5’ GAC CGA GGT GAA GTG C 3’ <span style="font-size: 12pt; font-family: 'Times New Roman','serif';">4GreenB 5’ GTT GTG TGG A GCA CTT CAG CTG TAG GTT CGT GGG TGA GCT ATC GTG GCT TCC TCG GTC 3’ <span style="font-size: 12pt; font-family: 'Times New Roman','serif';"> <span style="font-size: 12pt; font-family: 'Times New Roman','serif';">C’Tile <span style="font-size: 12pt; font-family: 'Times New Roman','serif';">4RedC 5’GGC AAT CCA CAA CCGG C 3’ <span style="font-size: 12pt; font-family: 'Times New Roman','serif';">4YellowC 5’ TCC ACA CAA C GCG GTT GTC CAA CTT ACC AGA TCC ACA AGC CGA CGT TAC AGG ATT GCC 3’ <span style="font-size: 12pt; font-family: 'Times New Roman','serif';">4BlueC 5’GTA TGG CGA ACG GTG TAG AGC CAA GA 3’ <span style="font-size: 12pt; font-family: 'Times New Roman','serif';">4GreenC 5’ GCT CTA CAG CAT CTG GTA AGT TGG TGT AAC GTC GGC TTG TCC GTT CGC 3’ <span style="font-size: 12pt; font-family: 'Times New Roman','serif';">D Tile <span style="font-size: 12pt; font-family: 'Times New Roman','serif';">4RedD 5’ CAT TCT GGA CGC CAT AAGA ATA GCA CCT CGA CTC ATT TGC CTG CGG TAG 3’ <span style="font-size: 12pt; font-family: 'Times New Roman','serif';">4YellowD 5’ TCA CTC TAC CGC ACC AGA ATG 3’ <span style="font-size: 12pt; font-family: 'Times New Roman','serif';">4BlueD 5’ CAG TAG CCT GCT ATC TTA TGG CGT GGC AAA TGA GTC GAG GAC GGA TCG 3’ <span style="font-size: 12pt; font-family: 'Times New Roman','serif';">4GreenD 5’ CAT ACC GAT CCG TGG CTA CTG 3’ <span style="font-size: 12pt; line-height: 200%; font-family: 'Times New Roman','serif';"> To create the DNA solution, 5 µl of each of the 16 oligonucleotides (short portions of the 16 dsDNA) were individually mixed with 45 µl of dH2O in 16 separate Eppendorff tubes. These tubes were then stored in a freezer until further use. To begin, a vortex was employed to liquefy each oligonucleotide solution. To create the stock solution, then, 5 µl of each of the 16 vortexed oligonucleotide solutions were added to 20 µl of Mg 2+/TAE (Tri-acetate) 10x buffer (which balanced the pH of the negative solution) and to 100 µl of 18 mega ohm (de-ionized) H2O. <span style="font-size: 12pt; line-height: 200%; font-family: 'Times New Roman','serif';">After the stock solution was created, it was annealed in the thermal cycler for 12 hours. This procedure was executed overnight and was placed in the thermal cycler by my mentor. The thermal cycler heats the solution to 90 degrees Celsius, and then cools it to 20 degrees Celsius, over the course of 12 hours. The annealing process allows for the complementary strands to bind together, via the polymerase chain reaction (PCR) process. <span style="font-size: 12pt; line-height: 200%; font-family: 'Times New Roman','serif';">When the annealing process was complete, 2 µl of the stock solution and 8 µl of the Mg 2+/TAE (1x) buffer were pippetted into an Eppendorff tube, for a total volume of 10 µl. This mixture was gently mixed by tapping the tube with a finger. The remaining stock solution was stored in a refrigerator if it was going to be used within 1-3 days. If not, then it was placed in the freezer. <span style="font-size: 12pt; line-height: 200%; font-family: 'Times New Roman','serif';">Next, the approximately 1 cm2 mica tile was cleaned by peeling off two surface layers with single-sided tape, a process termed “cleaving” the mica. After this, it was “vigorously” rinsed (i.e. rinsed with about 20 drops of 18 megaohm deionized H2O) and then dried completely with nitrogen gas. <span style="font-size: 12pt; line-height: 200%; font-family: 'Times New Roman','serif';">Next, the 10 µl of the DNA solution was deposited onto the surface, with a deposition time of 3 minutes. After the allotted time, the sample was “vigorously” rinsed with 18 mega ohm water and dried thoroughly with nitrogen gas. The sample was then placed on a puck that had a piece of double-sided tape attached to it. Next, the sample was viewed under the AFM in tapping mode. When an area appeared to have DNA rafts, images were captured of a 5µm2, a 2 µm2, and a 1 µm2 region at 256 pixels. The scan rate varied between 1.246-1.299 Hz/second. <span style="font-size: 12pt; line-height: 200%; font-family: 'Times New Roman','serif';">If, however, the sample was not going to be viewed the same day it was created, it was placed in a desiccator until use. The desiccator helps to prevent moisture-induced contaminants. <span style="font-size: 12pt; font-family: 'Times New Roman','serif';"> **<span style="font-size: 12pt; font-family: 'Times New Roman','serif';">Results ** <span style="font-size: 12pt; font-family: 'Times New Roman','serif';"> <span style="font-size: 12pt; line-height: 200%; font-family: 'Times New Roman','serif';">The results are divided into three major areas: identifying the right concentration of DNA and solution properties to produce the rafts, conducting section analysis to ensure that the particles on the sample are rafts, and plotting the number of DNA chains v. the number of DNA rafts per chain. <span style="font-size: 12pt; line-height: 200%; font-family: 'Times New Roman','serif';">First, obtaining the right mixture of DNA and the buffer solution was a matter of trial and error. Initally, the stock solution contained 80 µl of DNA, 10 µl of Mg2+/TAE (10x) buffer, and 10 µl of 18 mega ohm water. The resulting images under the AFM produced networks and web-like structures, and no clear images of DNA were identified. In essence, the solution was too concentrated, even when the buffer solution was added and the deposition time decreased. Images such as the following were obtained. <span style="font-size: 12pt; line-height: 200%; font-family: 'Times New Roman','serif';">A new, diluted stock solution, a 0.05mM solution, was then created which resulted in the elimination of the large clumps of DNA. Unfortunately, a new and different problem arose with this change: the formation of network and web-like structures on the sample surface. Some examples are presented in the following images. <span style="font-size: 11pt; font-family: 'Calibri','sans-serif'; mso-fareast-font-family: Calibri; mso-bidi-font-family: 'Times New Roman'; mso-ansi-language: EN-US; mso-fareast-language: EN-US; mso-bidi-language: AR-SA;"> <span style="font-size: 12pt; font-family: 'Times New Roman','serif'; mso-fareast-font-family: Calibri; mso-ansi-language: EN-US; mso-fareast-language: EN-US; mso-bidi-language: AR-SA;">Eventually, these networks were eliminated by rinsing the mica with 18 mega ohm water and drying it completely in the cleaning process before the deposition of DNA. So, from this point onward, every mica sample was “vigorously” rinsed with water before DNA deposition. The next variable that was altered was the mixture of different levels of DNA and the buffer in a homogenous solution before deposition on the mica surface. <span style="font-size: 12pt; line-height: 200%; font-family: 'Times New Roman','serif';">After obtaining this image, and seeing structures resembling DNA chains, or necklaces, I decided to dilute the solution further, with hopes to spread the molecules farther apart. Therefore, for the next set of samples, I made a new 0.05 mM stock solution, and created the samples using 2 µl of the new stock solution and 8 µl of the buffer. The following images were obtained <span style="font-size: 12pt; line-height: 200%; font-family: 'Times New Roman','serif';"> <span style="font-size: 12pt; line-height: 200%; font-family: 'Times New Roman','serif';">These images yielded much more promising results. Therefore, further analysis could be conducted to discover formation patterns in the DNA. <span style="font-size: 12pt; line-height: 200%; font-family: 'Times New Roman','serif';">In order to verify that the particles in these images were, indeed, DNA rafts, section analysis was conducted on the lengths and heights of the DNA rafts using the software for the AFM. Length and height measurements were conducted on 102 rafts. The sum of the horizontal measurements was a total of ~3780 nm. This was then divided by 102 to obtain ~37 nm, which is the accepted length of a DNA raft.9 The vertical distance measurements observed were ~132 nm, when, divided by 102, equals ~1.3 nm, which is within the range of the accepted height.9 <span style="font-size: 12pt; line-height: 200%; font-family: 'Times New Roman','serif';">Upon confirming the accuracy of the observations through comparing my observations with accepted measurements, I was able complete the third stage of the project: to plot the number of DNA raft chains v. the number of DNA rafts per chain by manually counting the number of rafts in a chain. This is illustrated by magnifying, every image obtained, so that a portion of it appeared as follows: <span style="font-size: 12pt; line-height: 200%; font-family: 'Times New Roman','serif';"> <span style="font-size: 12pt; line-height: 200%; font-family: 'Times New Roman','serif';"> <span style="font-size: 12pt; line-height: 200%; font-family: 'Times New Roman','serif';"> In this way, it was much easier to count the number of rafts in a chain accurately. In this image, for example, the number of rafts under the black line can be distinguished as 6 individual rafts. <span style="font-size: 12pt; line-height: 200%; font-family: 'Times New Roman','serif';"> The histogram for the aggregated data is shown below in Figure 7 from 5 images. <span style="font-size: 12pt; line-height: 200%; font-family: 'Times New Roman','serif';"> <span style="font-size: 12pt; font-family: 'Times New Roman','serif';">This suggests that DNA raft patterning is favorable to creating smaller chains, with between 1-4 rafts per chain. As the number of rafts per chain increases, the number of chains decrease, indicating that some factor interferes with the creation of larger chains. **<span style="font-size: 12pt; font-family: 'Times New Roman','serif';">Discussion ** <span style="font-size: 12pt; line-height: 200%; font-family: 'Times New Roman','serif';"> <span style="font-size: 12pt; line-height: 200%; font-family: 'Times New Roman','serif';">The first two goals of this study were to determine the most favorable conditions for the formation of DNA rafts on mica. The critical factors for successful deposition of DNA and raft formation were the creation of a diluted solution and a modified chip cleaning procedures, and low external influences when using the AFM. <span style="font-size: 12pt; line-height: 200%; font-family: 'Times New Roman','serif';">The rafts obtained were then analyzed and histograms obtained. These were compatible with the idea that the DNA rafts follow a patterning called “step-polymerization.” <span style="font-size: 12pt; line-height: 200%; font-family: 'Times New Roman','serif';"> <span style="font-size: 12pt; line-height: 200%; font-family: 'Times New Roman','serif';">The Carothers Equation, mentioned earlier, explains the relationship between the degree of conversion in the reaction and the average chain length in a sample. The higher the degree of conversion, the higher the predicted average chain length.8 The method of obtaining the degree of conversion is explained below. Since the original Carothers Equation produced a number that was outside a rational average, the modified equation was used because it takes into account whether one of the “sticky-ends” was not functioning. The notion that the rafts had a mono-functional component was supported with the Polydispersity Index (PDI) explained below. <span style="font-size: 12pt; line-height: 200%; font-family: 'Times New Roman','serif';">This PDI describes the spread of molecular mass in a sample containing polymers. All values are greater than 1, but values approach 1 if the polymer chains are of uniform length.8 This is not necessarily the case with these data, by fitting the data into the equation, PDI = Mw/Mn, where Mw= {Mo (1+P)}/ (1-P) and is the weight average molecular weight, and Mn = Mo/(1-P) is the number average molecular weight. 8 “P” in the equation is the extent of reaction, as in the Carothers’ Equation.8 The extent of the reaction, in turn, equals the number of monomers that reacted to form chains divided by the total number of monomers in a data sample. Mathematically, this is expressed as (No-N)/No, where No the total number of monomers in a data sample, and N is the number of un-reacted monomers.8 <span style="font-size: 12pt; line-height: 200%; font-family: 'Times New Roman','serif';">From the data, it is evident that the number of rafts peaks between 2-3 DNA Rafts per chain. When an average of the chain length is taken, however, the result is about 3.53 rafts per chain. Given this, then, when the data is plugged into the Carothers Equation, where chain length, Xn = 1/(1-P), where “P” is the extent of reaction, the result should be close to this average.8 However, the result is an average of 20.54 rafts per chain, when the degree of conversion is 95.1%. Therefore, another equation must be used, and in this case, the modified Carothers Equation for step polymerization with only a mono-functional component was used. 8 Using the modified equation, then, of Xn = {1+ (39/801)}/{1-(762/801)+(39/801)} yields an average chain length of 10.77 rafts/chain. <span style="font-size: 12pt; line-height: 200%; font-family: 'Times New Roman','serif';">The PDI for these data equals 1.951, which is close to a value of 2 that indicates step polymerization.8 Step polymerization is the condition of having multifunctional components that have an equal chance of reacting**.** 8 In this case, there are two “sticky-ends” that have an equal chance of reacting with other monomers or chains. It is irrelevant whether one “sticky-end” is already bonded to a chain; the other “sticky-end” has an equal chance of binding to either a monomer or another chain. 8 <span style="font-size: 12pt; line-height: 200%; font-family: 'Times New Roman','serif';">So, one of the main questions that arose is why my predicted chain length using the modified Carothers Equation for step-growth polymerization with a mono-functional unit was much higher than the average chain length, which came out to be ~3.53 rafts/chain, or about 4 rafts/chain. If this number is instead put in place of Xn in the equation, than (1-P) will be designated as “x” in order to find what the number of monomers in the data would have been. So, the equation looks like the following: 3.53 = (1+x)/{1-(1-x)+x}, where “x” comes to be ~0.165. So, to find the number of un-reacting monomers, multiply 0.165 by 801, which yields approximately 132 monomers. That is a difference of 93 monomers from the data obtained. <span style="font-size: 12pt; line-height: 200%; font-family: 'Times New Roman','serif';">Reasons for this discrepancy are usually due to defective rafts. Given the yield obtained, the predicted average chain length is higher than the average chain length. Therefore, there could have been “sticky-ends” that were deactivated in some way, either through bacteria that may have damaged the rafts or through decomposition of the monomer. Since DNA is a biological material, it is subject to many external forces that may inhibit self-assembly or functionality.10 <span style="font-size: 12pt; line-height: 200%; font-family: 'Times New Roman','serif';">Another possible reason for this discrepancy may be due to inability to count the number of rafts in every chain on a sample’s image. Some of the chains had indistinguishable rafts, and therefore could not be complied into the aggregate data. The data, then, may have had a higher average chain length and may have matched more closely to the predicted value from the Carothers Equation. <span style="font-size: 12pt; line-height: 200%; font-family: 'Times New Roman','serif';">Initially, the data was thought to follow chain-growth polymerization, which is the most common for polymers. Chain-growth polymerization occurs on an active site on a growing polymer. It also yields a PDI of between 10 and 20.8 However, since the PDI of the data yielded a value close to 2, the idea of chain-growth polymerization was eliminated. <span style="font-size: 12pt; font-family: 'Times New Roman','serif';"> **<span style="font-size: 12pt; line-height: 200%; font-family: 'Times New Roman','serif';">Conclusions ** <span style="font-size: 12pt; line-height: 200%; font-family: 'Times New Roman','serif';"> <span style="font-size: 12pt; line-height: 200%; font-family: 'Times New Roman','serif';">Collectively, the procedures and data indicate that DNA rafts can be produced, that they can be subjected to analysis, and that the analysis indicates step-growth polymerization. The production of DNA rafts is fairly straightforward, although ensuring the lack of defective rafts is difficult. This is an obstacle that must be overcome before any analysis can take place. <span style="font-size: 12pt; line-height: 200%; font-family: 'Times New Roman','serif';"> Regarding the analysis, once it can be certain that there are little or no defective rafts, the analysis will have to show that the degree of conversion for polymerization is similar. <span style="font-size: 12pt; line-height: 200%; font-family: 'Times New Roman','serif';">Lastly, the data indicate step-growth polymerization, which, because of the DNA’s two reactive sites, proves harder to determine what the spread of the chain length will be. The ideal situation would have chain-growth polymerization because in the creation of circuit boards, and the utilization of DNA rafts as a platform, having one active site where the other rafts fall into place would be easier to predict and mass produce. Therefore, if step-growth is the mode of polymerization for these specific rafts, greater care must be taken to evaluate whether the rafts are defective or not, in order to mass produce DNA platforms for nanotechnology. However, step-polymerization could be used to produce chains with an arbitrary number of rafts, such as the production of only “dimers” (2-raft chains). Nevertheless, the obstacle of defective rafts must be surmounted first, and degrees of conversion for polymerization must be similar before the predicted chain length of polymers using the Carothers Equation can be used to map DNA scaffolds for nanotechnology. <span style="font-size: 12pt; line-height: 200%; font-family: 'Times New Roman','serif';"> <span style="font-size: 12pt; line-height: 200%; font-family: 'Times New Roman','serif';"> <span style="font-size: 12pt; line-height: 200%; font-family: 'Times New Roman','serif';"> <span style="font-size: 12pt; line-height: 200%; font-family: 'Times New Roman','serif';">