RNA Internal Standard Synthesis by Nucleic Acid Sequence-Based Amplification for Competitive Quantitative Amplification Reactions

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• 作者：Wan-Yu Lo ; Antje J. Baeumner
• 刊名：Analytical Chemistry
• 出版年：2007
• 出版时间：February 15, 2007
• 年：2007
• 卷：79
• 期：4
• 页码：1548 - 1554
• 全文大小：235K
• 年卷期：v.79,no.4(February 15, 2007)
• ISSN：1520-6882

文摘

Nucleic acid sequence-based amplification (NASBA) reactions have been demonstrated to successfully synthesizenew sequences based on deletion and insertion reactions.Two RNA internal standards were synthesized for use incompetitive amplification reactions in which quantitativeanalysis can be achieved by coamplifying the internalstandard with the wild type sample. The sequences werecreated in two consecutive NASBA reactions using theE. coli clpB mRNA sequence as model analyte. Theprimer sequences of the wild type sequence were maintained, and a 20-nt-long segment inside the ampliconregion was exchanged for a new segment of similar GCcontent and melting temperature. The new RNA sequencewas thus amplifiable using the wild type primers anddetectable via a new inserted sequence. In the firstreaction, the forwarding primer and an additional 20-nt-long sequence was deleted and replaced by a new 20-nt-long sequence. In the second reaction, a forwardingprimer containing as 5' overhang sequence the wild typeprimer sequence was used. The presence of pure internalstandard was verified using electrochemiluminescenceand RNA lateral-flow biosensor analysis. Additional sequence deletion in order to shorten the internal standardamplicons and thus generate higher detection signals wasfound not to be required. Finally, a competitive NASBAreaction between one internal standard and the wild typesequence was carried out proving its functionality. Thisnew rapid construction method via NASBA providesadvantages over the traditional techniques since it requires no traditional cloning procedures, no thermocyclers, and can be completed in less than 4 h.