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style="background:#FFFFFF;text-align:left;text-indent:0pt;vertical-align:baseline;"> <span style="color:#333333;font-family:微软雅黑;font-size:10.5pt;font-style:normal;"> </span></p><p style="background:#FFFFFF;text-align:left;text-indent:0pt;vertical-align:baseline;"> <b><span style="background:#FFFFFF;color:#4476C1;font-family:微软雅黑;font-size:10.5pt;font-style:normal;font-weight:bold;"><span>一、</span>PCR引物设计原则</span></b></p><p style="background:#FFFFFF;text-align:left;text-indent:0pt;vertical-align:baseline;"> <span style="color:#333333;font-family:微软雅黑;font-size:10.5pt;font-style:normal;"> </span></p><p style="background:#FFFFFF;text-align:left;text-indent:0pt;vertical-align:baseline;"> <b><span style="background:#FFFFFF;color:#333333;font-family:微软雅黑;font-size:10.5pt;font-style:normal;font-weight:bold;">1、引物长度一般在15-30bp</span></b></p><p style="background:#FFFFFF;text-align:left;text-indent:0pt;vertical-align:baseline;"> <span style="background:#FFFFFF;color:#333333;font-family:微软雅黑;font-size:10.5pt;font-style:normal;"><span>引物长度一般为</span>15-30bp，常用的为18-27bp，但不应大于38bp，因为过长会导致其延伸温度大于74℃，不适于Taq DNA聚合酶进行反应；</span></p><p style="background:#FFFFFF;text-align:left;text-indent:0pt;vertical-align:baseline;"> <span style="color:#333333;font-family:微软雅黑;font-size:10.5pt;font-style:normal;"> </span></p><p style="background:#FFFFFF;text-align:left;text-indent:0pt;vertical-align:baseline;"> <b><span style="background:#FFFFFF;color:#333333;font-family:微软雅黑;font-size:10.5pt;font-style:normal;font-weight:bold;">2、引物GC含量一般为40%-60%</span></b></p><p style="background:#FFFFFF;text-align:left;text-indent:0pt;vertical-align:baseline;"> <span style="background:#FFFFFF;color:#333333;font-family:微软雅黑;font-size:10.5pt;font-style:normal;"><span>引物</span>GC含量一般为40%-60%，以45-55%为宜，GC含量过高或过低都不利于引发反应。上下游引物GC含量和Tm值要保持接近；</span></p><p style="background:#FFFFFF;text-align:left;text-indent:0pt;vertical-align:baseline;"> <span style="color:#333333;font-family:微软雅黑;font-size:10.5pt;font-style:normal;"> </span></p><p style="background:#FFFFFF;text-align:left;text-indent:0pt;vertical-align:baseline;"> <b><span style="background:#FFFFFF;color:#333333;font-family:微软雅黑;font-size:10.5pt;font-style:normal;font-weight:bold;">3、引物所对应的模板序列的Tm值最好在72℃左右</span></b></p><p style="background:#FFFFFF;text-align:left;text-indent:0pt;vertical-align:baseline;"> <span style="background:#FFFFFF;color:#333333;font-family:微软雅黑;font-size:10.5pt;font-style:normal;">Tm值在72℃左右可使复性条件最佳，至少要在55-80℃之间。Tm值的计算有多种方法，如按公式Tm＝4(G+C)＋2(A+T)，在Oligo软件中使用的是最邻近法(the nearest neighbor method)；</span></p><p style="background:#FFFFFF;text-align:left;text-indent:0pt;vertical-align:baseline;"> <span style="color:#333333;font-family:微软雅黑;font-size:10.5pt;font-style:normal;"> </span></p><p style="background:#FFFFFF;text-align:left;text-indent:0pt;vertical-align:baseline;"> <b><span style="background:#FFFFFF;color:#333333;font-family:微软雅黑;font-size:10.5pt;font-style:normal;font-weight:bold;">4、引物3’端的碱基一般不用A</span></b></p><p style="background:#FFFFFF;text-align:left;text-indent:0pt;vertical-align:baseline;"> <span style="background:#FFFFFF;color:#333333;font-family:微软雅黑;font-size:10.5pt;font-style:normal;"><span>引物</span>3’端的碱基一般不用A，因为A在错误引发位点的引发效率相对比较高。</span></p><p style="background:#FFFFFF;text-align:left;text-indent:0pt;vertical-align:baseline;"> <b><span style="background:#FFFFFF;color:#333333;font-family:微软雅黑;font-size:10.5pt;font-style:normal;font-weight:bold;">5、引物3’端出现3个以上的连续碱基，如GGG或CCC，也会使错误引发机率增加。</span></b></p><p style="background:#FFFFFF;text-align:left;text-indent:0pt;vertical-align:baseline;"> <span style="color:#333333;font-family:微软雅黑;font-size:10.5pt;font-style:normal;"> </span></p><p style="background:#FFFFFF;text-align:left;text-indent:0pt;vertical-align:baseline;"> <b><span style="background:#FFFFFF;color:#333333;font-family:微软雅黑;font-size:10.5pt;font-style:normal;font-weight:bold;">6、引物3’端的互补、二聚体或发夹结构也可能导致PCR反应失败。</span></b></p><p style="background:#FFFFFF;text-align:left;text-indent:0pt;vertical-align:baseline;"> <span style="color:#333333;font-family:微软雅黑;font-size:10.5pt;font-style:normal;"> </span></p><p style="background:#FFFFFF;text-align:left;text-indent:0pt;vertical-align:baseline;"> <b><span style="background:#FFFFFF;color:#333333;font-family:微软雅黑;font-size:10.5pt;font-style:normal;font-weight:bold;">7、如扩增编码区域，引物3’端不要终止于密码子的第3位，因密码子的第三位易发生简并，会影响扩增特异性与效率；</span></b></p><p style="background:#FFFFFF;text-align:left;text-indent:0pt;vertical-align:baseline;"> <span style="color:#333333;font-family:微软雅黑;font-size:10.5pt;font-style:normal;"> </span></p><p style="background:#FFFFFF;text-align:left;text-indent:0pt;vertical-align:baseline;"> <b><span style="background:#FFFFFF;color:#333333;font-family:微软雅黑;font-size:10.5pt;font-style:normal;font-weight:bold;">8、引物5’端可以修饰</span></b></p><p style="background:#FFFFFF;text-align:left;text-indent:0pt;vertical-align:baseline;"> <span style="background:#FFFFFF;color:#333333;font-family:微软雅黑;font-size:10.5pt;font-style:normal;"><span>引物</span>5’端序列对PCR影响不太大，因此常用来引进修饰位点或标记物。</span></p><p style="background:#FFFFFF;text-align:left;text-indent:0pt;vertical-align:baseline;"> <span style="color:#333333;font-family:微软雅黑;font-size:10.5pt;font-style:normal;"> </span></p><p style="background:#FFFFFF;text-align:left;text-indent:0pt;vertical-align:baseline;"> <b><span style="background:#FFFFFF;color:#333333;font-family:微软雅黑;font-size:10.5pt;font-style:normal;font-weight:bold;">9、碱基要随机分布，且引物自身和引物之间不能有连续4个碱基的互补。</span></b></p><p style="background:#FFFFFF;text-align:left;text-indent:0pt;vertical-align:baseline;"> <span style="background:#FFFFFF;color:#333333;font-family:微软雅黑;font-size:10.5pt;font-style:normal;"><span>引物序列在模板内应当没有相似性较高，尤其是</span>3’端相似性较高的序列，否则容易导致错误引发。降低引物与模板相似性的一种方法是，引物中四种碱基的分布最好是随机的，不要有聚嘌呤或聚嘧啶的存在。引物自身不应存在互补序列，否则引物自身会折叠成发夹结构（Hairpin）使引物本身复性。这种二级结构会因空间位阻而影响引物与模板的复性结合。引物自身不能有连续4个碱基的互补。两引物之间也不应具有互补性，尤其应避免3’端的互补重叠以防止引物二聚体（Dimer与Cross Dimer）的形成。引物之间不能有连续4个碱基的互补。引物二聚体及发夹结构如果不可避免的话，应尽量使其△G值不要过高（应小于4.5kcal/mol）。否则易导致引物二聚体带的产生，并且降低引物有效浓度而使PCR反应不能正常进行。</span></p><p style="background:#FFFFFF;text-align:left;text-indent:0pt;vertical-align:baseline;"> <span style="color:#333333;font-family:微软雅黑;font-size:10.5pt;font-style:normal;"> </span></p><p style="background:#FFFFFF;text-align:left;text-indent:0pt;vertical-align:baseline;"> <b><span style="background:#FFFFFF;color:#333333;font-family:微软雅黑;font-size:10.5pt;font-style:normal;font-weight:bold;">10、引物的ΔG值最好呈正弦曲线形状，即3’端ΔG值较低，而5’端和中间ΔG值相对较高。</span></b></p><p style="background:#FFFFFF;text-align:left;text-indent:0pt;vertical-align:baseline;"> <span style="background:#FFFFFF;color:#333333;font-family:微软雅黑;font-size:10.5pt;font-style:normal;">ΔG值（自由能）反应了引物与模板结合的强弱程度，一般情况下，引物的ΔG值最好呈正弦曲线形状，即3’端ΔG值较低，而5’端和中间ΔG值相对较高。3'端的ΔG值相对要低，且绝对值不要超过9，引物的3’端的ΔG值过高，容易在错配位点形成双链结构并引发DNA聚合反应。</span></p><p style="background:#FFFFFF;text-align:left;text-indent:0pt;vertical-align:baseline;"> <span style="color:#333333;font-family:微软雅黑;font-size:10.5pt;font-style:normal;"> </span></p><p style="background:#FFFFFF;text-align:left;text-indent:0pt;vertical-align:baseline;"> <span style="background:#FFFFFF;color:#333333;font-family:微软雅黑;font-size:10.5pt;font-style:normal;">11、引物应在核酸保守区内设计并具有特异性。引物与非特异扩增序列的同源性不要超过70%或有连续8个互补碱基同源。</span></p><p style="background:#FFFFFF;text-align:left;text-indent:0pt;vertical-align:baseline;"> <span style="color:#333333;font-family:微软雅黑;font-size:10.5pt;font-style:normal;"> </span></p><p style="background:#FFFFFF;text-align:left;text-indent:0pt;vertical-align:baseline;"> <b><span style="background:#FFFFFF;color:#4476C1;font-family:微软雅黑;font-size:10.5pt;font-style:normal;font-weight:bold;"><span>二、</span>Real Time PCR引物设计原则</span></b></p><p style="background:#FFFFFF;text-align:left;text-indent:0pt;vertical-align:baseline;"> <span style="color:#333333;font-family:微软雅黑;font-size:10.5pt;font-style:normal;"> </span></p><p style="background:#FFFFFF;text-align:left;text-indent:0pt;vertical-align:baseline;"> <span style="background:#FFFFFF;color:#333333;font-family:微软雅黑;font-size:10.5pt;font-style:normal;">Real Time PCR用引物与普通PCR引物设计要求不同。RealTime PCR是让目的基因按照理论值进行扩增，对非特异性反应和引物二聚体要求严格。</span></p></li> <li class="n_back"><a href="javascript:history.go(-1);">返回列表</a> </li> </ul></div> </div> </div> </div> </div> </div> <div id="foot"> <div class="foot"> <div class="wrap"> <div class="foot_left"> <div class="foot_b"> <div class="foot_p"> 产品&服务</div> <ul class="foot_ul"><li><a href="/web/20220129084805/http://husbio.com/products/23.aspx">ELISA试剂盒</a></li> <li><a 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