我司PCR实验的原理说明

<p class="MsoNormal"><span style="background-color: rgb(153, 204, 0);"><strong><span style="font-family: 宋体; font-size: 9pt;">PCR</span><span style="font-family: 宋体; font-size: 9pt;">的</span><span style="font-family: 宋体; font-size: 9pt;">原理</span></strong></span><span style="mso-spacerun:'yes';font-family:宋体;mso-ascii-font-family:Calibri;mso-hansi-font-family:Calibri;mso-bidi-font-family:'Times New Roman';font-size:9.0000pt;mso-font-kerning:1.0000pt;"><o:p></o:p></span></p> <p class="MsoNormal"><span style="mso-spacerun:'yes';font-family:宋体;mso-ascii-font-family:Calibri;mso-hansi-font-family:Calibri;mso-bidi-font-family:'Times New Roman';font-size:9.0000pt;mso-font-kerning:1.0000pt;">DNA<font face="宋体">的半保留复制是生物进化和传代的重要途径。双链</font><font face="Calibri">DNA</font><font face="宋体">在多种酶的作用下可以变性解旋成单链，在</font><font face="Calibri">DNA</font><font face="宋体">聚合酶的参与下，根据碱基互补配对原则复制成同样的两分子拷贝。在实验中发现，</font><font face="Calibri">DNA</font><font face="宋体">在高温时也可以发生变性解链，当温度降低后又可以复性成为双链。因此，通过温度变化控制</font><font face="Calibri">DNA</font><font face="宋体">的变性和复性，加入设计引物，</font><font face="Calibri">DNA</font><font face="宋体">聚合酶、</font><font face="Calibri">dNTP</font><font face="宋体">就可以完成特定基因的体外复制。</font></span><span style="mso-spacerun:'yes';font-family:宋体;mso-ascii-font-family:Calibri;mso-hansi-font-family:Calibri;mso-bidi-font-family:'Times New Roman';font-size:9.0000pt;mso-font-kerning:1.0000pt;"><o:p></o:p></span></p> <p class="MsoNormal"><span style="mso-spacerun:'yes';font-family:宋体;mso-ascii-font-family:Calibri;mso-hansi-font-family:Calibri;mso-bidi-font-family:'Times New Roman';font-size:9.0000pt;mso-font-kerning:1.0000pt;"><font face="宋体">但是，</font>DNA<font face="宋体">聚合酶在高温时会失活，因此，每次循环都得加入新的</font><font face="Calibri">DNA</font><font face="宋体">聚合酶，不仅操作烦琐，而且价格昂贵，制约了</font><font face="Calibri">PCR</font><font face="宋体">技术的应用和发展。</font></span><span style="mso-spacerun:'yes';font-family:宋体;mso-ascii-font-family:Calibri;mso-hansi-font-family:Calibri;mso-bidi-font-family:'Times New Roman';font-size:9.0000pt;mso-font-kerning:1.0000pt;"><o:p></o:p></span></p> <p class="MsoNormal"><span style="mso-spacerun:'yes';font-family:宋体;mso-ascii-font-family:Calibri;mso-hansi-font-family:Calibri;mso-bidi-font-family:'Times New Roman';font-size:9.0000pt;mso-font-kerning:1.0000pt;"><font face="宋体">耐热</font>DNA<font face="宋体">聚合酶</font><font face="Calibri">--Taq</font><font face="宋体">酶的发现对于</font><font face="Calibri">PCR</font><font face="宋体">的应用有里程碑的意义，该酶可以耐受</font><font face="Calibri">90</font><font face="宋体">℃以上的高温而不失活，不需要每个循环加酶，使</font><font face="Calibri">PCR</font><font face="宋体">技术变得非常简捷、同时也大大降低了成本，</font><font face="Calibri">PCR</font><font face="宋体">技术得以大量应用，并逐步应用于临床。</font></span><span style="mso-spacerun:'yes';font-family:宋体;mso-ascii-font-family:Calibri;mso-hansi-font-family:Calibri;mso-bidi-font-family:'Times New Roman';font-size:9.0000pt;mso-font-kerning:1.0000pt;"><o:p></o:p></span></p> <p class="MsoNormal"><span style="mso-spacerun:'yes';font-family:宋体;mso-ascii-font-family:Calibri;mso-hansi-font-family:Calibri;mso-bidi-font-family:'Times New Roman';font-size:9.0000pt;mso-font-kerning:1.0000pt;">PCR<font face="宋体">技术的基本原理类似于</font><font face="Calibri">DNA</font><font face="宋体">的天然复制过程，其特异性依赖于与靶序列两端互补的寡核苷酸引物。</font><font face="Calibri">PCR</font><font face="宋体">由变性</font><font face="Calibri">--</font><font face="宋体">退火</font><font face="Calibri">--</font><font face="宋体">延伸三个基本反应步骤构成：</font></span><span style="mso-spacerun:'yes';font-family:宋体;mso-ascii-font-family:Calibri;mso-hansi-font-family:Calibri;mso-bidi-font-family:'Times New Roman';font-size:9.0000pt;mso-font-kerning:1.0000pt;"><o:p></o:p></span></p> <p class="MsoNormal"><span style="color: rgb(255, 0, 0);"><strong><span style="font-family: 宋体; font-size: 9pt;">①&nbsp;</span></strong></span><span style="mso-spacerun:'yes';font-family:宋体;mso-ascii-font-family:Calibri;mso-hansi-font-family:Calibri;mso-bidi-font-family:'Times New Roman';font-size:9.0000pt;mso-font-kerning:1.0000pt;">模板<font face="Calibri">DNA</font><font face="宋体">的变性：模板</font><font face="Calibri">DNA</font><font face="宋体">经加热至</font><font face="Calibri">93</font><font face="宋体">℃左右一定时间后，使模板</font><font face="Calibri">DNA</font><font face="宋体">双链或经</font><font face="Calibri">PCR</font><font face="宋体">扩增形成的双链</font><font face="Calibri">DNA</font><font face="宋体">解离，使之成为单链，以便它与引物结合，为下轮反应作准备；</font></span><span style="mso-spacerun:'yes';font-family:宋体;mso-ascii-font-family:Calibri;mso-hansi-font-family:Calibri;mso-bidi-font-family:'Times New Roman';font-size:9.0000pt;mso-font-kerning:1.0000pt;"><o:p></o:p></span></p> <p class="MsoNormal"><span style="color: rgb(255, 0, 0);"><strong><span style="font-family: 宋体; font-size: 9pt;">②&nbsp;</span></strong></span><span style="mso-spacerun:'yes';font-family:宋体;mso-ascii-font-family:Calibri;mso-hansi-font-family:Calibri;mso-bidi-font-family:'Times New Roman';font-size:9.0000pt;mso-font-kerning:1.0000pt;">模板<font face="Calibri">DNA</font><font face="宋体">与引物的退火（复性）：模板</font><font face="Calibri">DNA</font><font face="宋体">经加热变性成单链后，温度降至</font><font face="Calibri">55</font><font face="宋体">℃左右，引物与模板</font><font face="Calibri">DNA</font><font face="宋体">单链的互补序列配对结合；</font></span><span style="mso-spacerun:'yes';font-family:宋体;mso-ascii-font-family:Calibri;mso-hansi-font-family:Calibri;mso-bidi-font-family:'Times New Roman';font-size:9.0000pt;mso-font-kerning:1.0000pt;"><o:p></o:p></span></p> <p class="MsoNormal"><span style="color: rgb(255, 0, 0);"><strong><span style="font-family: 宋体; font-size: 9pt;">③</span></strong></span><strong><span style="mso-spacerun:'yes';font-family:宋体;mso-ascii-font-family:Calibri;mso-hansi-font-family:Calibri;mso-bidi-font-family:'Times New Roman';font-size:9.0000pt;mso-font-kerning:1.0000pt;">&nbsp;</span></strong><span style="mso-spacerun:'yes';font-family:宋体;mso-ascii-font-family:Calibri;mso-hansi-font-family:Calibri;mso-bidi-font-family:'Times New Roman';font-size:9.0000pt;mso-font-kerning:1.0000pt;">引物的延伸：<font face="Calibri">DNA</font><font face="宋体">模板</font><font face="Calibri">--</font><font face="宋体">引物结合物在</font><font face="Calibri">72</font><font face="宋体">℃、</font><font face="Calibri">DNA</font><font face="宋体">聚合酶（如</font><font face="Calibri">TaqDNA</font><font face="宋体">聚合酶）的作用下，以</font><font face="Calibri">dNTP</font><font face="宋体">为反应原料，靶序列为模板，按碱基互补配对与半保留复制原理，合成一条新的与模板</font><font face="Calibri">DNA</font><font face="宋体">链互补的半保留复制链，重复循环变性</font><font face="Calibri">--</font><font face="宋体">退火</font><font face="Calibri">--</font><font face="宋体">延伸三过程就可获得更多的&ldquo;半保留复制链&rdquo;，而且这种新链又可成为下次循环的模板。每完成一个循环需</font><font face="Calibri">2</font><font face="宋体">～</font><font face="Calibri">4</font><font face="宋体">分钟，</font><font face="Calibri">2</font><font face="宋体">～</font><font face="Calibri">3</font><font face="宋体">小时就能将待扩目的基因扩增放大几百万倍。</font></span><span style="mso-spacerun:'yes';font-family:宋体;mso-ascii-font-family:Calibri;mso-hansi-font-family:Calibri;mso-bidi-font-family:'Times New Roman';font-size:9.0000pt;mso-font-kerning:1.0000pt;"><o:p></o:p></span></p>