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Oligo(dT) primer generates a high frequency of truncated cDNAs through internal poly(A) priming during reverse transcription

机译:Oligo(dT)引物在逆转录过程中通过内部poly(A)引发产生高频率的截短的cDNA

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摘要

We have analyzed a systematic flaw in the current system of gene identification: the oligo(dT) primer widely used for cDNA synthesis generates a high frequency of truncated cDNAs through internal poly(A) priming. Such truncated cDNAs may contribute to 12% of the expressed sequence tags in the current dbEST database. By using a synthetic transcript and real mRNA templates as models, we characterized the patterns of internal poly(A) priming by oligo(dT) primer. We further demonstrated that the internal poly(A) priming can be effectively diminished by replacing the oligo(dT) primer with a set of anchored oligo(dT) primers for reverse transcription. Our study indicates that cDNAs designed for genomewide gene identification should be synthesized by use of the anchored oligo(dT) primers, rather than the oligo(dT) primers, to diminish the generation of truncated cDNAs caused by internal poly(A) priming.
机译:我们分析了当前基因鉴定系统中的一个系统缺陷:广泛用于cDNA合成的oligo(dT)引物通过内部poly(A)引发产生高频率的截短cDNA。此类截短的cDNA可能占当前dbEST数据库中12%的表达序列标签的比重。通过使用合成的成绩单和真实的mRNA模板作为模型,我们表征了由oligo(dT)引物内部poly(A)引发的模式。我们进一步证明,可以通过用一组锚定的oligo(dT)引物代替oligo(dT)引物来进行反转录,来有效地减少内部poly(A)引发。我们的研究表明,应通过使用锚定的oligo(dT)引物而不是oligo(dT)引物来合成专为全基因组基因鉴定而设计的cDNA,以减少内部poly(A)引发引起的截短cDNA的产生。

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