Characteristics of selective polymerase chain reaction (PCR) using two‐base anchored primers and improvement of its specificity
Characteristics of selective polymerase chain reaction (PCR) using two‐base anchored primers and improvement of its specificity
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二碱基锚定引物选择性聚合酶链式反应（PCR）的特点及其特异性的提高
DOI：
10.1002/elps.1150191805
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发表时间：
1998
期刊：
影响因子：
2.9
通讯作者：
H. Kambara
中科院分区：
文献类型：
--
作者：
K. Okano;C. Uematsu;H. Matsunaga;H. Kambara
我们开发了一种可靠的方法，用于消除选择性聚合酶链反应（PCR）中的碱基错配扩增，该方法用于扩增限制性内切酶反应产生的未知序列片段。所提出的程序应用扩增片段长度多态性（AFLP）高保真。选择性PCR利用已知的聚合酶反应特性，即互补链延伸受到模板与引物3′-末端匹配的强烈影响。然而，经常观察到假阳性扩增，因为用于区分片段的末端碱基（通常，1-3个锚序列）的特异性不足以分离每个片段。因此，研究了用于选择性PCR分离每个片段的方案。在引物3 ′端第4个碱基位置人工引入单碱基错配，以提高引物3′端锚定2碱基的杂交特异性。PCR反应在66°C下进行以防止假阳性扩增。具有AA、AT、TA和TT锚定碱基序列的引物的浓度必须比其他引物的浓度大三倍，因为这些序列的Tm值低于其他序列。由于该方法能将所有片段高保真地分组，因此可应用于基于AFLP的基因组序列差异分析和基因发现。
We have developed a reliable method for eliminating base‐mispair amplification in selective polymerase chain reaction (PCR), which is utilized for amplifying unknown sequence fragments produced by restriction enzyme reaction. The proposed procedure applies amplified fragment length polymorphism (AFLP) with high fidelity. Selective PCR utilizes the known polymerase reaction characteristic that the complementary strand extension is strongly affected by matching a template with the 3′‐terminus of the primers. However, false positive amplification is frequently observed because the specificity of terminal bases for discrimination of fragments (usually, 1–3 anchor sequences) is not enough to separate each fragment. A protocol for the selective PCR separation of every fragment was therefore investigated. A single‐base mismatch was artificially introduced on the 4th base position from the 3′ end of the primers to improve the hybridization specificity of anchored 2‐bases at the 3′ termini of primers. PCR reaction was carried out at 66°C to prevent false positive amplification. The concentration of the primers having anchored‐base sequences of AA, AT, TA, and TT must be three times larger than that of other primers because the Tm values for these sequences are lower than the others. As all the fragments can be separated into groups with high fidelity, the improved selective PCR will be applied to gene finding and analyzing differences on genome sequences based on AFLP.
影响因子：
14.9
作者：
HUANG, MM;ARNHEIM, N;GOODMAN, MF
通讯作者：
GOODMAN, MF
影响因子：
14.9
作者：
LIANG, P;AVERBOUKH, L;PARDEE, AB
通讯作者：
PARDEE, AB