Parallel Overlap Assembly Research Articles

Comparison of Material Consumption, Experimental Protocols and Computation Time in DNA Computing

One of the major constraints in DNA computation is the exponential increase in material consumption and computation time for larger computation size in DNA computing particularly in critical stages such as initial pool generation and extraction during gel electrophoresis. In DNA computation, both the hybridization-ligation method and parallel overlap assembly method can be utilized to generate the initial pool of all possible solutions. In this paper, we discuss and compare the implementation of N × N Boolean matrix multiplication via in vitro implementation between Hybridization-Ligation Method and Parallel Overlap Assembly Method to show that selection of tools and protocols affect the cost effectiveness of a computation in terms of the material consumption, protocol steps and execution time to compute. In general, the the parallel overlap assembly method performs better than hybridization-ligation method in terms of the three parameters mentioned. The calculations are based on approximation of unique sequence strands required for the computation and not actual calculations on the nmol concentration.

Comparison of different methods to analyze a DNA computing library using the polymerase chain reaction

Screening and analysis of collections of DNA molecules is a standard aspect of many DNA computing approaches. We describe the use of three different polymerase chain reaction (PCR) detection methods to screen specific members of a 5-site, 2-variable DNA computing library previously created using parallel overlap assembly of unique sequences generated from the SynDCode program. The three PCR methods (conventional gel-based PCR, SYBR Green real-time PCR and TaqMan real-time PCR) could all successfully identify individual library members separately or in a mixture. The TaqMan approach was also able to identify members in the original library we had not yet sequenced, providing more evidence supporting our hypothesis that the DNA library we generated may be complete. We expect these three approaches will be useful in future screening of other DNA computing libraries and structures.

A Simulation Software for DNA Computing Algorithms Implementation

The capturing of gel electrophoresis image represents the output of a DNA computing algorithm. Before this image is being captured, DNA computing involves parallel overlap assembly (POA) and polymerase chain reaction (PCR) that is the main of this computing algorithm. However, the design of the DNA oligonucleotides to represent a problem is quite complicated and is prone to errors. In order to reduce these errors during the design stage before the actual in-vitro experiment is carried out; a simulation software capable of simulating the POA and PCR processes is developed. This simulation software capability is unlimited where problem of any size and complexity can be simulated, thus saving cost due to possible errors during the design process. Information regarding the DNA sequence during the computing process as well as the computing output can be extracted at the same time using the simulation software.

Experimental implementation and analysis of a DNA computing readout method based on real-time PCR with TaqMan probes

A new readout approach for the Hamiltonian Path Problem (HPP) in DNA computing based on the real-time polymerase chain reaction (PCR) is experimentally implemented and analyzed. Several types of fluorescent probes and detection mechanisms are currently employed in real-time PCR, including SYBR Green, molecular beacons, and hybridization probes. In this study, real-time amplification performed using the TaqMan probes is adopted, as the TaqMan detection mechanism can be exploited for the design and development of the proposed readout approach. Double-stranded DNA molecules of length 140 base-pairs are selected as the input molecules, which represent the solving path for an HPP instance. These input molecules are prepared via the self-assembly of 20-mer and 30-mer single-stranded DNAs, by parallel overlap assembly. The proposed readout approach consists of two steps: real-time amplification in vitro using TaqMan-based real-time PCR, followed by information processing in silico to assess the results of real-time amplification, which in turn, enables extraction of the Hamiltonian path. The performance of the proposed approach is compared with that of conventional graduated PCR. Experimental results establish the superior performance of the proposed approach, relative to graduated PCR, in terms of implementation time.

Hybridization-Ligation Versus Parallel Overlap Assembly: An Experimental Comparison of Initial Pool Generation for Direct-Proportional Length-Based DNA Computing

Previously, direct-proportional length-based DNA computing (DPLB-DNAC) for solving weighted graph problems has been reported. The proposed DPLB-DNAC has been successfully applied to solve the shortest path problem, which is an instance of weighted graph problems. The design and development of DPLB-DNAC is important in order to extend the capability of DNA computing for solving numerical optimization problem. According to DPLB-DNAC, after the initial pool generation, the initial solution is subjected to amplification by polymerase chain reaction and, finally, the output of the computation is visualized by gel electrophoresis. In this paper, however, we give more attention to the initial pool generation of DPLB-DNAC. For this purpose, two kinds of initial pool generation methods, which are generally used for solving weighted graph problems, are evaluated. Those methods are hybridization-ligation and parallel overlap assembly (POA). It is found that for DPLB-DNAC, POA is better than that of the hybridization-ligation method, in terms of population size, generation time, material usage, and efficiency, as supported by the results of actual experiments.

In Vitro Implementation of k-shortest Paths Computation with Graduated PCR

In this paper, an in vitro implementation of DNA computing for solving k-shortest paths problem of a weighted graph is reported. The encoding is designed in such a way that every path is encoded by oligonucleotides and the length of the path is directly proportional to the length of oligonucleotides. For initial pool generation, parallel overlap assembly is employed for efficient generation of all candidate answers. After the initial solution is subjected to amplification by polymerase chain reaction (PCR), k-shortest paths could be visualized by polyacrylamide gel electrophoresis (PAGE) and the selection can be done. The visualization of the output, in fact, relies on the appearance of DNA bands on a gel image. Further, it is shown that a method called graduated PCR is a good subsequent bio-molecular reaction for obtaining molecular information hidden in the output DNA. Graduated PCR is also crucial to prove the correctness of the in vitro computation. The experimental results show the effectiveness of the proposed DNA-based computation and prove that the k-shortest paths problem has been successfully solved on a DNA computer.

State transitions by molecules

In our previous paper, we described a method by which a state machine is implemented by a single-stranded DNA molecule whose 3′-end sequence encodes the current state of the machine. Successive state transitions are performed in such a way that the current state is annealed onto an appropriate portion of DNA encoding the transition table of the state machine and the next state is copied to the 3′-end by extension with polymerase. In this paper, we first show that combined with parallel overlap assembly, a single series of successive transitions can solve NP-complete problems. This means that the number of necessary laboratory steps is independent from the problem size. We then report the results of two experiments concerning the implementation of our method. One is on isothermal reactions which greatly increase the efficiency of state transitions compared with reactions controlled by thermal cycles. The other is on the use of unnatural bases for avoiding out-of-frame annealing. The latter result can also be applied to many DNA-based computing paradigms.

Parallel Overlap Assembly for the Construction of Computational DNA Libraries

Algorithms for computing with DNA currently require the construction of pools of molecules in which each distinct molecule represents a different starting point for the calculation. We have begun building such pools using the technique of parallel overlap assembly that is already used for the generation of diversity in biologically useful combinatorial search techniques such as gene shuffling. Unlike these applications, a pool in a molecular computer must be complete, containing all possible strands, and ordered, having minimal contamination from incorrectly assembled DNA. We present an experiment in which parallel overlap assembly is used to construct a computational pool and an experiment in which this pool is used to solve the NP-complete maximal-clique problem.