我司PCR實(shí)驗(yàn)的原理說明

<p class="MsoNormal"><span style="background-color: rgb(153, 204, 0);"><strong><span style="font-family: 宋體; font-size: 9pt;">PCR</span><span style="font-family: 宋體; font-size: 9pt;">的</span><span style="font-family: 宋體; font-size: 9pt;">原理</span></strong></span><span style="mso-spacerun:'yes';font-family:宋體;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:宋體;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="宋體">的半保留復(fù)制是生物進(jìn)化和傳代的重要途徑。雙鏈</font><font face="Calibri">DNA</font><font face="宋體">在多種酶的作用下可以變性解旋成單鏈，在</font><font face="Calibri">DNA</font><font face="宋體">聚合酶的參與下，根據(jù)堿基互補(bǔ)配對原則復(fù)制成同樣的兩分子拷貝。在實(shí)驗(yàn)中發(fā)現(xiàn)，</font><font face="Calibri">DNA</font><font face="宋體">在高溫時(shí)也可以發(fā)生變性解鏈，當(dāng)溫度降低后又可以復(fù)性成為雙鏈。因此，通過溫度變化控制</font><font face="Calibri">DNA</font><font face="宋體">的變性和復(fù)性，加入設(shè)計(jì)引物，</font><font face="Calibri">DNA</font><font face="宋體">聚合酶、</font><font face="Calibri">dNTP</font><font face="宋體">就可以完成特定基因的體外復(fù)制。</font></span><span style="mso-spacerun:'yes';font-family:宋體;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:宋體;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="宋體">但是，</font>DNA<font face="宋體">聚合酶在高溫時(shí)會(huì)失活，因此，每次循環(huán)都得加入新的</font><font face="Calibri">DNA</font><font face="宋體">聚合酶，不僅操作煩瑣，而且價(jià)格昂貴，制約了</font><font face="Calibri">PCR</font><font face="宋體">技術(shù)的應(yīng)用和發(fā)展。</font></span><span style="mso-spacerun:'yes';font-family:宋體;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:宋體;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="宋體">耐熱</font>DNA<font face="宋體">聚合酶</font><font face="Calibri">--Taq</font><font face="宋體">酶的發(fā)現(xiàn)對于</font><font face="Calibri">PCR</font><font face="宋體">的應(yīng)用有里程碑的意義，該酶可以耐受</font><font face="Calibri">90</font><font face="宋體">℃以上的高溫而不失活，不需要每個(gè)循環(huán)加酶，使</font><font face="Calibri">PCR</font><font face="宋體">技術(shù)變得非常簡捷、同時(shí)也大大降低了成本，</font><font face="Calibri">PCR</font><font face="宋體">技術(shù)得以大量應(yīng)用，并逐步應(yīng)用于臨床。</font></span><span style="mso-spacerun:'yes';font-family:宋體;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:宋體;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="宋體">技術(shù)的基本原理類似于</font><font face="Calibri">DNA</font><font face="宋體">的天然復(fù)制過程，其特異性依賴于與靶序列兩端互補(bǔ)的寡核苷酸引物。</font><font face="Calibri">PCR</font><font face="宋體">由變性</font><font face="Calibri">--</font><font face="宋體">退火</font><font face="Calibri">--</font><font face="宋體">延伸三個(gè)基本反應(yīng)步驟構(gòu)成：</font></span><span style="mso-spacerun:'yes';font-family:宋體;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: 宋體; font-size: 9pt;">①&nbsp;</span></strong></span><span style="mso-spacerun:'yes';font-family:宋體;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="宋體">的變性：模板</font><font face="Calibri">DNA</font><font face="宋體">經(jīng)加熱至</font><font face="Calibri">93</font><font face="宋體">℃左右一定時(shí)間后，使模板</font><font face="Calibri">DNA</font><font face="宋體">雙鏈或經(jīng)</font><font face="Calibri">PCR</font><font face="宋體">擴(kuò)增形成的雙鏈</font><font face="Calibri">DNA</font><font face="宋體">解離，使之成為單鏈，以便它與引物結(jié)合，為下輪反應(yīng)作準(zhǔn)備；</font></span><span style="mso-spacerun:'yes';font-family:宋體;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: 宋體; font-size: 9pt;">②&nbsp;</span></strong></span><span style="mso-spacerun:'yes';font-family:宋體;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="宋體">與引物的退火（復(fù)性）：模板</font><font face="Calibri">DNA</font><font face="宋體">經(jīng)加熱變性成單鏈后，溫度降至</font><font face="Calibri">55</font><font face="宋體">℃左右，引物與模板</font><font face="Calibri">DNA</font><font face="宋體">單鏈的互補(bǔ)序列配對結(jié)合；</font></span><span style="mso-spacerun:'yes';font-family:宋體;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: 宋體; font-size: 9pt;">③</span></strong></span><strong><span style="mso-spacerun:'yes';font-family:宋體;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:宋體;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="宋體">模板</font><font face="Calibri">--</font><font face="宋體">引物結(jié)合物在</font><font face="Calibri">72</font><font face="宋體">℃、</font><font face="Calibri">DNA</font><font face="宋體">聚合酶（如</font><font face="Calibri">TaqDNA</font><font face="宋體">聚合酶）的作用下，以</font><font face="Calibri">dNTP</font><font face="宋體">為反應(yīng)原料，靶序列為模板，按堿基互補(bǔ)配對與半保留復(fù)制原理，合成一條新的與模板</font><font face="Calibri">DNA</font><font face="宋體">鏈互補(bǔ)的半保留復(fù)制鏈，重復(fù)循環(huán)變性</font><font face="Calibri">--</font><font face="宋體">退火</font><font face="Calibri">--</font><font face="宋體">延伸三過程就可獲得更多的&ldquo;半保留復(fù)制鏈&rdquo;，而且這種新鏈又可成為下次循環(huán)的模板。每完成一個(gè)循環(huán)需</font><font face="Calibri">2</font><font face="宋體">～</font><font face="Calibri">4</font><font face="宋體">分鐘，</font><font face="Calibri">2</font><font face="宋體">～</font><font face="Calibri">3</font><font face="宋體">小時(shí)就能將待擴(kuò)目的基因擴(kuò)增放大幾百萬倍。</font></span><span style="mso-spacerun:'yes';font-family:宋體;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>