LoPPS: A long PCR product sequencing method for rapid characterisation of long amplicons

被引：10

作者：

Emonet, Sebastien ^{[1
]}

Grard, Gilda ^{[1
]}

Brisbarre, Nadege ^{[1
]}

Moureau, Gregory ^{[1
]}

Temmam, Sarah ^{[1
]}

Charrel, Remi ^{[1
]}

de Lamballerie, Xavier ^{[1
]}

机构：

[1] Unite Virus Emergents, EA3292, IFR48, IRD,UR034,Fac Med, F-13005 Marseille, France

来源：

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS | 2006年 / 344卷 / 04期

关键词：

sequencing; PCR; RNA viruses; LoPPS;

D O I：

10.1016/j.bbrc.2006.04.015

中图分类号：

Q5 [生物化学]; Q7 [分子生物学];

学科分类号：

071010 ; 081704 ;

摘要：

Here, we propose an optimised protocol (LoPPS, long PCR product sequencing) which allows the fast, cost-attractive, and high-throughput sequencing of long PCR products. LoPPS constitutes an alternative to the primer-walking technology which is expensive and time consuming but remains the current standard procedure. It is based on the ultrasonic shearing, polishing, and cloning of PCR or RT-PCR products and is compatible with 96- or 384-well microplate systems in which bacterial growth, preparation of plasmid DNA, and sequencing can be automated. We present results obtained from 24 different RT-PCR products (2.5-4.8 kbp long) obtained from various RNA viruses and fully sequenced using LoPPS. The method proved to be robust and fast. It was successfully used on a low amount of DNA and allowed each target nucleotide position to be controlled twice or more, with a final cost which is one-third of that of primer-walking. (c) 2006 Elsevier Inc. All rights reserved.

引用

页码：1080 / 1085

页数：6

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