PCR amplification of an Escherichia coli gene using mixed primers containing deoxyinosine at ambiguous positions in degenerate amino acid codons.
PCR amplification of an Escherichia coli gene using mixed primers containing deoxyinosine at ambiguous positions in degenerate amino acid codons.
复制标题
使用在简并氨基酸密码子的模糊位置含有脱氧肌苷的混合引物对大肠杆菌基因进行 PCR 扩增。
DOI：
10.1093/nar/18.10.3080
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发表时间：
1990
影响因子：
14.9
通讯作者：
Dekker,EE
中科院分区：
文献类型：
--
作者：
Patil,RV;Dekker,EE
聚合酶链式反应(PCR)是一种强大的技术，广泛用于从少量DNA中进行特定序列的酶扩增；该过程由一对寡核苷酸引物指导，该引物位于待扩增DNA片段的相反链的两侧并杂交(1)。在针对未知序列的DNA的情况下，代表所有密码子选择的混合引物是利用DNA编码的蛋白质的氨基酸序列合成的。尽管混合引物的聚合酶链式反应在各种条件下都是成功的，但特异扩增的程度主要取决于是否使用足够的引物和退火点的选择。混合引物中可能的结构数量可能非常多，因此，对于含有大量简并密码子的蛋白质来说，可能变得无效。这一问题可以通过合成在不明确位置含有脱氧肌苷的混合引物来解决。在这里，我们成功地用两个分别对应于酶的10个N端和10个C端氨基酸的混合引物，成功地扩增了大肠杆菌2-酮-4-羟基戊二酸醛缩酶的编码区，该酶的10个N-端和10个C-端的氨基酸在不明确的位置含有脱氧肌苷，在5‘端有EcoR1接头。N端和C端分别含有4个和7个脱氧肌苷残基。使用的是未经高效液相色谱纯化的引物。经30次循环后的DNA产物经琼脂糖凝胶电泳法分离后，在55℃的温度下有一条预期大小的主带(当温度低于55℃时出现两条带)。将扩增的DNA亚克隆到pUC18的EcoRI区，测序所得的氨基酸序列与已知的醛缩酶的一级结构完全一致。这种方法通常对具有高度简并密码子的蛋白质有用。图1显示了经琼脂糖凝胶(0.7%)电泳法分离和溴化乙锭染色后的PCR产物。50-Al-PCR反应混合物含有0.5，T4G的大肠杆菌基因组DNA，每个引物1.0jig，每个核苷酸0.2 mM，美国生化公司的Taq DNA聚合酶0.5/11(2.5U)，在100 mM Tris HCl缓冲液(pH 8.3)中，含有500 mM KCl，15 mM MgCl2和0.1%(w/v)明胶。反应混合物上覆盖100升矿物油，以避免蒸发。每个循环在94℃下1分钟，在55℃下1分钟，缓慢上升(在2分钟内)到72℃，然后在72℃下2分钟，由Perkin-Elmer CETUS的热循环仪调节。通道1：DNA大小标记。通道2和3：30个循环后，在反应混合物的2，11中扩增产物；箭头表示从缩醛酶编码区扩增的DNA。
The polymerase chain reaction (PCR) is a powerful technique widely used for enzymatic amplification of specific sequences from small amounts of DNA; the process is directed by a pair of oligonucleotide primers which flank and hybridize to opposite strands of the DNA segment to be amplified (1). In cases of targeting DNA of unknown sequence, mixed primers representing all codon choices are synthesized using the amino acid sequence of the protein encoded by the DNA. Although PCR with mixed primers is clearly successful under a wide range of conditions, the degree ofspecific amplification depends critically on the use of sufficient primer and the choice of annealing temperature. The number of possible structures in mixed primers can be very large and, as a consequence, could become ineffective for proteins with numerous degenerate codons. This problem can be solved by synthesizing mixed primers having deoxyinosine at ambiguous positions. We show here successful PCR amplification of the coding region of 2-keto-4-hydroxyglutarate aldolase of Escherichia coli (2) using two mixed primers corresponding to the 10 N-terminal and the 10 C-terminal amino acids of the enzyme containing deoxyinosine at ambiguous positions and the EcoRl linker at the 5'ends. The N-terminal and the C-terminal primer contained 4 and 7 deoxyinosine residues, respectively. The primers were used without HPLC purification. PCR products obtained after 30 cycles, which were separated by agarose gel electrophoresis, showed a single major DNA band of the predicted size when primers were annealed at 55 C (two bands were seen when the annealing temperature was below 55 C). The amino acid sequence deduced from sequencing the PCR-amplified DNA subcloned into the EcoRI site of pUC18 plasmid was found to be in complete agreement with the known primary structure of the aldolase (2). This method is useful generally for proteins with highly degenerate codons. Figure 1 shows the PCR products after separation by agarose gel (0.7%) electrophoresis and stained with ethidium bromide. A 50-Al PCR reaction mixture contained 0.5, t4g of E. coli genomic DNA, 1.0 jig of each primer, 0.2 mM of each nucleotide, 0.5/1l (2.5 U) of Taq DNA polymerase from US Biochemical Corp. in 100 mM Tris HCl buffer (pH 8.3) containing 500 mM KCl, 15 mM MgCl2, and 0.1%(w/v) gelatin. The reaction mixture was covered with 100 1l of mineral oil to avoid evaporation. Each cycle was 1 min at 94 C, 1 min at 55 C, slow rise (in 2 min) to 72 C, and then 2 min at 72 C regulated by a thermal cycler from Perkin-Elmer Cetus. Lane 1: DNA size markers. Lane 2 and 3: PCR products in 2, 1l of reaction mixture after 30 cycles; the arrow indicates the amplified DNA from the aldolase coding region.