Nam Douglas Kyung, Lee Sanggyu, Zhou Guolin, Cao Xiaohong, Wang Clarence, Clark Terry, Chen Jianjun, Rowley Janet D, Wang San Ming
Department of Medicine, Center for Functional Genomics, University of Chicago, 5841 South Maryland Avenue, MC2115, Chicago, IL 60637, USA.
Proc Natl Acad Sci U S A. 2002 Apr 30;99(9):6152-6. doi: 10.1073/pnas.092140899. Epub 2002 Apr 23.
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.
广泛用于cDNA合成的寡聚（dT）引物通过内部聚腺苷酸化引发产生高频截短cDNA。此类截短cDNA可能占当前dbEST数据库中表达序列标签的12%。通过使用合成转录本和真实mRNA模板作为模型，我们表征了寡聚（dT）引物内部聚腺苷酸化引发的模式。我们进一步证明，通过用一组用于逆转录的锚定寡聚（dT）引物替代寡聚（dT）引物，可有效减少内部聚腺苷酸化引发。我们的研究表明，为全基因组基因识别设计的cDNA应使用锚定寡聚（dT）引物而非寡聚（dT）引物进行合成，以减少由内部聚腺苷酸化引发导致的截短cDNA的产生。
