載脂蛋白CⅢ基因位于11號染色體與載脂蛋白AⅠ-AⅣ形成一基因族，它位于載脂蛋白AⅠ下游，有三個(gè)內(nèi)含子和四個(gè)外顯子。目前對載脂蛋白CⅢ基因3'末端非編碼區(qū)C-G對換產(chǎn)生一個(gè)Sac-Ⅰ酶切位點(diǎn)與冠心病的關(guān)系未有定論。用多聚酶鏈反應(yīng)(PCR)擴(kuò)增載脂蛋白CⅢ基因3'末端233bp，再用Sac-1酶切擴(kuò)增產(chǎn)物，可以檢測載脂蛋白CⅢ基因3'末端的突變及其與動(dòng)脈粥樣硬化相關(guān)性。本節(jié)采用PCR-RFLP檢測ApoCⅢ基因型。
一、儀器與試劑
1.儀器：電泳儀，基因擴(kuò)增儀。
2.試劑：TaqDNA聚合酶，dNTP，DNA參考物，Sca-1酶。
二、操作方法
1.模DNA提取
取用EDTA抗凝全血100μl加試劑(0.32mol/L蔗糖，5mmol/l MgCl2,1% TritonX-100,0.01mol/L Tris-HClpH7.6)，振搖至溶解均勻透明，3000r/min離心10min,棄上清，沉淀部分加0.9NaCl溶液洗一次，加預(yù)冷試劑Ⅱ(75mmol/l NaCl,24mmol/L EDTA)100μl，20%SDS15μl，蛋白酶K10μl(6g/L)于65℃水浴4h，加200μl酚和氯仿：異戊醇(24：1)各提兩次，上清加無水乙醇1.0ml放置于-20℃2h，以12kr/min離心5min，真空干燥后加50μlTE緩沖液溶解，4℃保存?zhèn)溆谩?/p>
2.多聚酶鏈反應(yīng)
Primer1：5'-GTG ACC GAT GGC TTC AGT TCC CTG-3'，primer2:5'-GGt AGG AGA GCA CTC AGA ATA CA-3'。反應(yīng)總體積為50μl，擴(kuò)增緩沖液所含物質(zhì)終濃度為：Tris-HCl，pH8.3，67mmol/L；MgCl225mmol/L；KCl 50mmol/L；明膠0.01%，dNTP各0.2～0.5μmol/L，模板約10μg，加40μl液體石蠟，94℃預(yù)變性4min，加Tag聚合酶1.5U，以93℃1min、65℃1.5min、72℃1.5min，循環(huán)30次。
3.擴(kuò)增產(chǎn)物酶切
取擴(kuò)增產(chǎn)物20μl加入NH4AC20μl，再加300μl無水乙醇混均置-20℃1h，10000r/min離心7min棄上清，干燥后加Sac-1酶液10μl37℃保溫2h。
4.擴(kuò)增產(chǎn)物堿基對分子量測定
采用GB公司產(chǎn)DNa marker,以已知DNA片段bp數(shù)相對應(yīng)的電泳遷移距離，取半對數(shù)作圖制成標(biāo)準(zhǔn)曲線，再根據(jù)等測定擴(kuò)增產(chǎn)物DNA片段移動(dòng)距離在標(biāo)準(zhǔn)曲線上求出其bp數(shù)。
三、結(jié)果分析
采用本節(jié)設(shè)計(jì)引物擴(kuò)增產(chǎn)物為ApoCⅢ基因3'非編碼區(qū)233bp，該產(chǎn)物經(jīng)Sac-1酶切可出現(xiàn)三種結(jié)果；①酶切后仍為一條帶，電泳遷移率未發(fā)生改變，為S1S1純合子；②酶切后除原始帶外又增加了兩條帶，查標(biāo)準(zhǔn)DNa marker曲線在158bp和75bp相同，為S2S2雜合子；③酶切后原始帶消失，出現(xiàn)兩條新帶，其電泳遷移率與158bp和75bp相同，為S2S2純合子。S2為擴(kuò)增的基因產(chǎn)物中C-G對換增加一個(gè)酶切位點(diǎn)。
檢查100名正常人中有S2雜合子28名，純合子2名；68名冠心病人中S2雜合16名，純合子4名，它們的頻率變化見表(19-4)。S2基因頻率兩組間無明顯差別。
表19-4 載脂蛋白CⅢ基因Sac-Ⅰ酶切頻率表
| 級別 | n | 基因型 | 頻 率 | |||
| S1S1 | S1S2 | S2S2 | S1 | S2 | ||
| 正常人 | 100 | 70 | 28 | 2 | 0.84 | 0.16 |
| 冠心病 | 68 | 48 | 16 | 4 | 0.82 | 0.176 |
X=0.079,p>0.05(兩組比較)
(徐瓊)
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