Controlled hot start and improved specificity in carrying out PCR utilizing touch-up and loop incorporated primers (TULIPS)
Controlled hot start and improved specificity in carrying out PCR utilizing touch-up and loop incorporated primers (TULIPS)
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DOI：
10.2144/00295st03
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发表时间：
2000-11-01
期刊：
影响因子：
2.7
通讯作者：
Silverman, M
中科院分区：
文献类型：
--
作者：
Ailenberg, M;Silverman, M
PCR技术通常产生非特异性产物。为了克服这一问题，设计了一种简单、特异、高效的方法：修饰环掺入引物（TULIPS）-PCR。该方法利用环引物（即，自退火至3'区并抑制聚合起始的另外的非模板5'序列）。反应加热后，引物熔化，引发热启动。该反应还使用修饰，预循环，退火温度逐渐升高，以确保正确的配对。该方法已被验证与甘油醛-3-磷酸脱氢酶（GAPD）引物，其一般适用性证明：特异性扩增的人明胶酶A转基因的基因组DNA extmcredfrorn转基因小鼠的尾巴。TULIPS-PCR方案是一种新的方法。与降落和手动热启动PCR相比，该方法中使用的自退火引物提供了改进的特异性和更稳健的合成。它的进行不需要打开、暂停或向反应混合物中加入任何非反应物组分，如蜡抗体或非特异性dsDNA。
The PCR technique often yields nonspecific products. To overcome this problem, a simple, specific and efficient method was de signed: touch-up, and loop incorporated primers (TULIPS)-PCR. This approach utilizes loop primers (i.e., additional nontemplate 5' sequence that self-anneals to the 3' region and inhibits initiation of polymerization). Upon heating of the reaction, the primers melt, initiating hot start. The reaction also uses touch-up, pre-cycling with gradual elevation in annealing temperatures to ensure correct pairing. The method has been validated with glyceraldehyde-3-phosphate dehydrogenase (GAPD) primers, and its general applicability is demonstrated by: specific amplification of the human gelatinase A transgene from genomic DNA extmcredfrorn transgenic mice tails. The TULIPS-PCR protocol is a novel method. The self-annealing primers utilized in this,method offer improved specificity and more robust synthesis compared with touch-down and manual hot start PCR. It is performed without the need to open, pause or add to the reaction mixture any nonreactant components, such as wax antibody or nonspecific dsDNA.