Mutations of the activation-associated phosphorylation sites at codons 308 and 473 of protein kinase B are absent in human melanoma
Mutations of the activation-associated phosphorylation sites at codons 308 and 473 of protein kinase B are absent in human melanoma
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DOI：
10.1007/s004030100236
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发表时间：
2001-07-01
影响因子：
3
通讯作者：
Deichmann, M
中科院分区：
文献类型：
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作者：
Waldmann, V;Wacker, J;Deichmann, M
V. Waldmann（美国）· J. Wacker· M.德国海德堡大学皮肤科戴希曼系2，69115 Heidelberg，德国电话：+ 49-6221-568505，传真：+ 49-6221-568509（见下文），扩增产物中密码子T 308为280 bp，密码子T 473为189 bp。由于可用于本分析的肿瘤材料的量低，我们决定应用"SMART“方案（在RNA模板的5 '端的转换机制; Clontech，Palo Alto，CA）。为了合成第一链，根据改良的硫氰酸胍方法[18]，使用马夸特等人描述的硅胶的选择性结合特性，制备60 - 170 ng肿瘤RNA。[19]（Qiagen，希尔登，德国）按照供应商的说明使用。如Clontech所述，在热循环仪中进行长距离PCR，包括在95 ℃下60秒，随后在95 ℃下5秒，在65 ℃下5秒，在68 ℃下6分钟，持续24个循环。在15、18、21和24个循环后，将5 μl反应混合物进行琼脂糖凝胶电泳，以确定适当的循环次数。该程序之后进行常规PCR，使用45个循环，95 ℃ 30秒，56 ℃ 30秒，72 ℃ 60秒。PCR扩增产物包括PKB/AKT基因T308密码子280 bp和T473密码子189 bp。通过琼脂糖凝胶电泳监测扩增产物的纯度和预期长度（图1）。扩增的特异性通过
V. Waldmann (И)· J. Wacker· M. Deichmann Department of Dermatology, University of Heidelberg, Voßstr. 2, 69115 Heidelberg, Germany Tel.:+ 49-6221-568505, Fax:+ 49-6221-568509 (see below) in amplification products of 280 bp for codon T 308 and of 189 bp for codon T 473. Due to the low amount of tumour material available for the present analysis, we decided to apply a „SMART “protocol (switching mechanism at 5′-end of RNA template; Clontech, Palo Alto, Calif.). For synthesis of the first strand, between 60 and 170 ng of tumour RNA, prepared according to a modified guanidinium thiocyanate method [18], using selective binding properties of a silica gel described by Marquardt et al.[19](Qiagen, Hilden, Germany) was used following the instructions of the supplier. A longdistance PCR, as described by Clontech, was carried out in a thermal cycler comprising 60 s at 95 C, followed by 5 s at 95 C, 5 s at 65 C, 6 min at 68 C for 24 cycles. After 15, 18, 21 and 24 cycles, 5 µl of the reaction mixture was subjected to agarose gel electrophoresis to determine an adequate cycle number. This procedure was followed by a conventional PCR using 45 cycles of 30 s at 95 C, 30 s at 56 C, and 60 s at 72 C. The PCR amplification products comprised sequences of 280 bp for codon T 308 and 189 bp for codon T 473 of PKB/AKT. The amplification products were monitored by agarose gel electrophoresis for purity and expected length (Fig. 1). The specificity of amplification was checked by