Single-molecule reverse transcription polymerase chain reaction using water-in-oil emulsion

被引:40
作者
Nakano, M [1 ]
Nakai, N [1 ]
Kurita, H [1 ]
Komatsu, J [1 ]
Takashima, K [1 ]
Katsura, S [1 ]
Mizuno, A [1 ]
机构
[1] Toyohashi Univ Technol, Dept Ecol Engn, Toyohashi, Aichi 4418580, Japan
基金
日本学术振兴会;
关键词
reverse transcription polymerase chain reaction; water-in-oil emulsion; single-molecule detection;
D O I
10.1263/jbb.99.293
中图分类号
Q81 [生物工程学(生物技术)]; Q93 [微生物学];
学科分类号
071005 ; 0836 ; 090102 ; 100705 ;
摘要
A new method based on a combination of reverse transcription polymerase chain reaction (RT-PCR) and a water-in-oil (W/O) emulsion was developed. Reverse transcription and initial thermal cycles were carried out in droplets of the W/O emulsion. Then, the droplets were united, followed by remaining secondary PCR cycles. This two-step method succeeded in detecting a single RNA molecule.
引用
收藏
页码:293 / 295
页数:3
相关论文
共 12 条
[1]   Single-molecule DNA amplification and analysis in an integrated microfluidic device [J].
Lagally, ET ;
Medintz, I ;
Mathies, RA .
ANALYTICAL CHEMISTRY, 2001, 73 (03) :565-570
[2]   RAPID AND SIMPLE PCR ASSAY FOR QUANTITATION OF HUMAN-IMMUNODEFICIENCY-VIRUS TYPE-1 RNA IN PLASMA - APPLICATION TO ACUTE RETROVIRAL INFECTION [J].
MULDER, J ;
MCKINNEY, N ;
CHRISTOPHERSON, C ;
SNINSKY, J ;
GREENFIELD, L ;
KWOK, S .
JOURNAL OF CLINICAL MICROBIOLOGY, 1994, 32 (02) :292-300
[3]   AMPLIFICATION AND DETECTION OF A SINGLE-MOLECULE OF HUMAN-IMMUNODEFICIENCY-VIRUS RNA [J].
NAKAMURA, S ;
KATAMINE, S ;
YAMAMOTO, T ;
FOUNG, SKH ;
KURATA, T ;
HIRABAYASHI, Y ;
SHIMADA, K ;
HINO, S ;
MIYAMOTO, T .
VIRUS GENES, 1993, 7 (04) :325-338
[4]   Single-step single-molecule PCR of DNA with a homo-priming sequence using a single primer and hot-startable DNA polymerase [J].
Nakano, H ;
Kobayashi, K ;
Ohuchi, S ;
Sekiguchi, S ;
Yamane, T .
JOURNAL OF BIOSCIENCE AND BIOENGINEERING, 2000, 90 (04) :456-458
[5]   Single-molecule PCR using water-in-oil emulsion [J].
Nakano, M ;
Komatsu, J ;
Matsuura, S ;
Takashima, K ;
Katsura, S ;
Mizuno, A .
JOURNAL OF BIOTECHNOLOGY, 2003, 102 (02) :117-124
[6]   In vitro method for the generation of protein libraries using PCR amplification of a single DNA molecule and coupled transcription/translation [J].
Ohuchi, S ;
Nakano, H ;
Yamane, T .
NUCLEIC ACIDS RESEARCH, 1998, 26 (19) :4339-4346
[7]   Armored RNA technology for production of ribonuclease-resistant viral RNA controls and standards [J].
Pasloske, BL ;
Walkerpeach, CR ;
Obermoeller, RD ;
Winkler, M ;
DuBois, DB .
JOURNAL OF CLINICAL MICROBIOLOGY, 1998, 36 (12) :3590-3594
[8]   Using RT-PCR and bDNA assays to measure non-clade BHIV-1 subtype RNA [J].
Pasquier, C ;
Sandres, K ;
Salama, G ;
Puel, J ;
Izopet, J .
JOURNAL OF VIROLOGICAL METHODS, 1999, 81 (1-2) :123-129
[9]   High-throughput, cloning-independent protein library construction by combining single-molecule DNA amplification with in vitro expression [J].
Rungpragayphan, S ;
Kawarasaki, Y ;
Imaeda, T ;
Kohda, K ;
Nakano, H ;
Yamane, T .
JOURNAL OF MOLECULAR BIOLOGY, 2002, 318 (02) :395-405
[10]   Man-made cell-like compartments for molecular evolution [J].
Tawfik, DS ;
Griffiths, AD .
NATURE BIOTECHNOLOGY, 1998, 16 (07) :652-656