Efficient polymerase chain reaction from single DNA molecules

2018 Fiscal Year Research-status Report

• Project/Area Number: 18K14260
• Research Institution: Japan Agency for Marine-Earth Science and Technology
• Principal Investigator: 張 翼 国立研究開発法人海洋研究開発機構, 海洋生命理工学研究開発センター, 研究員 (40795358)
• Project Period (FY): 2018-04-01 – 2021-03-31
• Keywords: PCR / Protein synthesis / Microfabrication / Micromanipulation / Single molecule / Cell-free

Outline of Annual Research Achievements

I established a work-flow for single-molecule DNA-initiated PCR reactions at an exclusive-use clean space in JAMSTEC. I applied this technique to recover single DNA molecules from a single femtoliter droplet where a single DNA molecule triggers cell-free transcription and translation reactions to synthesized its corresponding protein. I wrote and submitted a research paper to summarize this work. This paper passed the initial screening and the first round of review and is under the second round of revision.
As my system can synthesize proteins from a single DNA molecule, I applied this system as an orthogonal readout means to interrogate the performance of DNA-relevant enzymes. I am working on the preparation of another manuscript for this work.

Current Status of Research Progress

2: Research has progressed on the whole more than it was originally planned.

Reason

As JAMSTEC does not hold microfabrication facilities, I made my effort to establish a microfabrication space outside JAMSTEC with a relatively low running cost. This space is also open for my colleagues who intended to use my microdevices for our ongoing collaborations.
I did not only confirm the excellent performance of PCR for single DNA molecules at my newly established working space but also extended the application of my protein synthesis technique to a new research direction for the assessment of characteristics of DNA-related enzymes with unprecedented sensitivity.
During the period of the first year, I have already written two research papers under the support of this funding.

Strategy for Future Research Activity

I will push this study forward according to my initial plan and continue to explore the possibility of other extensions of my system like what I did in the first year.

Causes of Carryover

An intended expenditure of molecular biology reagents was saved by existing reagents in other laboratories. The combined money may be used for reagents, consumables, microfabrication, outsourcing services, and open access fees of publication.