Targeted rapid amplification of cDNA ends (T-RACE)--an improved RACE reaction through degradation of non-target sequences.
Targeted rapid amplification of cDNA ends (T-RACE)--an improved RACE reaction through degradation of non-target sequences.
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DOI：
10.1093/nar/gkq816
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发表时间：
2010-11
影响因子：
14.9
通讯作者：
Johnston IA
中科院分区：
文献类型：
--
作者：
Bower NI;Johnston IA
扩增cDNA的5′端，虽然理论上很简单，但往往很难实现。我们描述了一种特异性扩增cDNA末端的新方法。掺入含dUTP的PCR引物的寡聚dT衔接子引发掺入dUTP的第一链cDNA合成。使用Cap finder方法，将另一个不同的含有衔接子的dUTP添加到新合成的cDNA的3′端。通过PCR实现掺入dUTP的第二链合成，使用与掺入cDNA末端的衔接子序列互补的含dUTP的引物。含有双链cDNA的dUTP可作为任何基因3′或5′端特异性扩增的通用模板。为了扩增cDNA的末端，使用单一基因特异性引物和标准dNTP进行不对称PCR。纯化不对称PCR产物，并通过尿嘧啶DNA糖基化酶降解含有dUTP的非靶转录物，仅留下在不对称PCR期间产生的那些转录物。随后使用巢式基因特异性引物和3′或5′ T-RACE引物的PCR导致cDNA末端的特异性扩增。该方法可用于从单个cDNA合成反应中特异性扩增许多cDNA的3′和5′末端。
Amplification of the 5′ ends of cDNA, although simple in theory, can often be difficult to achieve. We describe a novel method for the specific amplification of cDNA ends. An oligo-dT adapter incorporating a dUTP-containing PCR primer primes first-strand cDNA synthesis incorporating dUTP. Using the Cap finder approach, another distinct dUTP containing adapter is added to the 3′ end of the newly synthesized cDNA. Second-strand synthesis incorporating dUTP is achieved by PCR, using dUTP-containing primers complimentary to the adapter sequences incorporated in the cDNA ends. The double-stranded cDNA-containing dUTP serves as a universal template for the specific amplification of the 3′ or 5′ end of any gene. To amplify the ends of cDNA, asymmetric PCR is performed using a single gene-specific primer and standard dNTPs. The asymmetric PCR product is purified and non-target transcripts containing dUTP degraded by Uracil DNA glycosylase, leaving only those transcripts produced during the asymmetric PCR. Subsequent PCR using a nested gene-specific primer and the 3′ or 5′ T-RACE primer results in specific amplification of cDNA ends. This method can be used to specifically amplify the 3′ and 5′ ends of numerous cDNAs from a single cDNA synthesis reaction.
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DOI：
10.1073/pnas.85.23.8998
发表时间：
1988-12-01
影响因子：
11.1
作者：
FROHMAN, MA;DUSH, MK;MARTIN, GR
通讯作者：
MARTIN, GR
影响因子：
2.7
作者：
Olivarius, Signe;Plessy, Charles;Carninci, Piero
通讯作者：
Carninci, Piero
影响因子：
14.9
作者：
Schmidt, W M;Mueller, M W
通讯作者：
Mueller, M W
影响因子：
14.9
作者：
Schramm, G;Bruchhaus, I;Roeder, T
通讯作者：
Roeder, T
影响因子：
7
作者：
Salehi-Ashtiani, Kourosh;Lin, Chenwei;Vidal, Marc
通讯作者：
Vidal, Marc