PCR-based gene synthesis with overlapping unisense-oligomers asymmetric extension supported by a simulator for oligonucleotide extension achieved 1 kbp dsDNA.

We formulated a method to synthesize 1 kbp DNA fragments using 'oligomer unidirectional joining method' via asymmetric extension supported by a simulator for oligonucleotide extension (AESOE). In this study, trials were conducted on 41 sets of different genomic pieces of ten flaviviral genomes, and 31 bacterial 16s rRNA fragments with sizes ranging from 500 bases to 1.0 kbp. Synthetic gene production was found to be successful in all those sets. The synthesis method has three steps: the first step is a seven-linked AESOE, the second step is the linking of the 400-base fragments from the first step, and the third step is the final amplification. Our present approach is highly reproducible and may no longer require optimization of oligomer design.

Prediction of PCR amplification from primer and template sequences using recurrent neural network.

We have developed a novel method to predict the success of PCR amplification for a specific primer set and DNA template based on the relationship between the primer sequence and the template. To perform the prediction using a recurrent neural network, the usual double-stranded formation between the primer and template nucleotide sequences was herein expressed as a five-lettered word. The set of words (pseudo-sentences) was placed to indicate the success or failure of PCR targeted to learn recurrent neural network (RNN). After learning pseudo-sentences, RNN predicted PCR results from pseudo-sentences which were created by primer and template sequences with 70% accuracy. These results suggest that PCR results could be predicted using learned RNN and the trained RNN could be used as a replacement for preliminary PCR experimentation. This is the first report which utilized the application of neural network for primer design and prediction of PCR results.

An improved gene synthesis method with asymmetric directions of oligonucleotides designed using a simulation program.

Artificial gene synthesis based on oligonucleotide augmentation is known as overlap extension PCR which generates a variety of intermediate synthetic products. The orientation and concentration of oligomers can be adjusted to reduce the synthesis of intermediates and optimize the full-length process of DNA synthesis, using a simulation program for serial oligomer extension. The efficiency of the serial oligomer extension process is predicted to be greatest when oligomers are in a 'forward-reverse-reverse-reverse' direction. Oligomers with such designed directions demonstrated generation of the desired product in the shortest time (number of cycles) by repeated annealing and elongation. This method, named Asymmetric Extension supported by a Simulator for Oligonucleotide Extension (AESOE), has shown efficiency and effectiveness with potentials for future improvements and optimal usage in DNA synthesis.