Development of technique for digital counting of DNA copy number at the single-cell level

2014 Fiscal Year Final Research Report

• Project/Area Number: 24360344
• Research Category: Grant-in-Aid for Scientific Research (B)
• Allocation Type: Partial Multi-year Fund
• Section: 一般
• Research Field: Biofunction/Bioprocess
• Research Institution: Waseda University
• Principal Investigator: TAKEYAMA Haruko 早稲田大学, 理工学術院, 教授 (60262234)
• Co-Investigator(Kenkyū-buntansha): YOKOTA Yasuko 国立感染症研究所, 免疫部, 室長 (20182701) ; HOSOKAWA Masahito 早稲田大学, ナノ・ライフ創新研究機構, 次席研究員(研究院助教) (60722981)
• Project Period (FY): 2012-04-01 – 2015-03-31
• Keywords: DNA / HIV / デジタルカウント / 一分子PCR / 単一細胞

Outline of Final Research Achievements

In this study, we aimed to develop a technique for digital counting of low copy viral DNA within infected cells. We developed a microfluidic device for generation of microdroplets for droplet-based digital PCR. In this method, the target genes were separately amplified within picoliter droplets and detected with fluorescent microscopy. The number of fluorescent droplets correlated with the copy numbers of target genes. In addition, we constructed lentivirus vector modeled after HIV. After optimization of the reaction conditions of droplet digital PCR, we demonstrated its highly sensitive detection of proviral DNA within a small number of infected cells during early infection process.

Free Research Field

プロセス工学