循环逆转录反应方法及应用条件研究
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国家自然科学基金资助项目(39700082).


Repeated Reverse Transcription Reaction (RRTR) Protocol and Its Application
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This work was supported by the National Natural Science Foundation of China(39700082).

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    摘要:

    建立了循环逆转录反应方法(repeated reverse transcription reaction, RRTR).其原理是通过控制高温变性-低温退火延伸的循环过程,反复利用起始RNA模板进行逆转录反应.RNA杂交的结果证明在RRTR过程中,RNA模板是保持稳定的;点杂交结果表明cDNA产物量得到了增加;定量PCR的结果表明RRTR是cDNA线性增长过程.结果提示,RRTR可有效地提高RNA检测灵敏度,并适合应用于定量分析等领域.

    Abstract:

    The RRTR can linearly increase cDNA products via repeated reverse transcription reactions controlled by temperatures and catalyzed by FD-TRT (FD thermostable reverse transcriptase). The stability of RNA in RRTR is proved via the Northern blot, and the increase in cDNA products along with that in reacting cycles is shown via the Dot hybridization. After components of the reaction system are optimized, the RRTR combined with PCR is applied in quantification of HCV RNA through changing the reacting cycles of the RRTR. It can be concluded that due to the linearity of RRTR, the RRTR is a convenient method to enhance the sensitivity and efficacy of RNA related study. It can be used in some fields which full-length cDNA is not required, such as quantification of RNA or cDNA, RACE (rapid amplification of cDNA end), and different display.

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陈燃,金詟,伍迪,唐榕,毛裕民.循环逆转录反应方法及应用条件研究[J].生物化学与生物物理进展,2001,28(4):560-562

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  • 收稿日期:2000-09-29
  • 最后修改日期:2000-12-12
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