High-throughput and high-fidelity automatic synthetic method for long-chain template-free deoxyribonucleic acid (DNA)

Abstract

The invention discloses a high-throughput and high-fidelity automatic synthetic method for a long-chain template-free deoxyribonucleic acid (DNA) and provides a method with an automatic liquid-removing workstation as a core. The method can finish automatic synthesis under extremely low human interference and even can automatically synthesize a DNA sequence of an eukaryotic gene group (10000,000 basepair). The lowest human interference is required by an automatic synthetic center and the whole synthesis process is performed under a sterile condition, so the pollution to a sample is reduced to the great extent. The automatic liquid-removing workstation can very precisely extract and release quantitative reagents, so errors caused by human operation are avoided, and the success rate is greatly improved. The automatic synthetic center is highly higher than human operation in operation speed and can work without stop at full speed, so the production efficiency is improved, simultaneously the labor and the time are reduced by reducing the errors, high-throughput and high-fidelity automatic synthesis on a large segment of DNA is realized, and the synthesis cost is greatly reduced.

Description

technical field [0001] The invention belongs to the field of DNA synthesis, and in particular relates to a high-throughput and high-fidelity automatic synthesis method of long-chain template-free DNA. Background technique [0002] In 1970, Agarwal and others spent 20 person-years to synthesize 75 base pairs (base pair, bp for short) (K. L. Agarwal, H. Buchi, M. H. Caruthers, N. Gupta, H. G. Khorana, K. Kleppe, A. Kumar, E. Ohtsuka, U. L. Rajbhandary, J. H. V. de Sande, V. Sgaramella, H. Weber, and T. Yamada. Total synthesis of the gene for an alanine transfer ribonucleic acid from yeast. Nature, 227(5253):27–34, Jul. 1970). Nowadays, with the advancement of chemical synthesis technology, DNA synthesis companies can synthesize sequences of thousands of base pairs at a relatively reasonable time and cost. However, the current synthesis technology mainly relies on manpower, and the error rate increases rapidly with the length of the synthesized sequence. Traditional molecula...

AI Technical Summary

Problems solved by technology

However, the current synthesis technology mainly relies on manpower, and the error rate increases rapidly with the length of the synthetic sequence.

Traditional molecular biology methods rely on the existence of natural DNA templates. The required DNA fragments are obtained by designing appropriate primers for PCR reactions, and then these fragments are spliced ​​together by ligation reactions. However, this traditional method is not suitable for template-free Synthetic DNA

In recent years, the Massachusetts Institute of Technology has proposed the BioBrick method, which is to place commonly used DNA fragments in a set of pre-defined enzyme cutting sites (so-called BioBricks), and each time two BiroBricks are cut with restriction endonucleases. Flattening, but a scar (scar) will be produced between the two BioBricks. It is conceivable that this method will produce a large number of scars for long-chain DNA, resulting in unpredictable phenotypic effects