專利名稱：：一種全血dna的提取方法
技術領域：
：本發明涉及一種全血DNA的提取方法。技術背景目前，提取全血DNA的方法一般為首先用紅細胞裂解液破碎紅細胞，然后通過離心使白細胞沉淀，然后反復洗滌以棄去血紅蛋白，雖然這樣能提取出純度較高的DNA，但是在破碎、離心過程中有相當數量的白細胞亦遭受破壞，尤其是凍存多年的陳舊血液；由于數量不等的白細胞隨著血紅蛋白一起被丟棄，因而造成了DNA的大量損失；有些血液樣本資源十分i貴，來源有限，不可再生，丟失會造成無可估量向的損失，其后果不可挽回。因此，急需一種既能保證純度、提取率又高的方法來提取全血DNA。
發明內容針對上述現有技術的不足，本發明提供了一種提取率高的全血DNA的提取方法。本發明是通過以下技術方案實現的-一種全血DNA高產出率的提取方法，包括以下步驟-(1).取凍融處理的ACD抗凝血，置于離心試管中，加入含有240ug/ml蛋白酶K的緩沖液，振蕩混勻，置于50'C水浴2028小時，水浴過程中緩慢搖動離心管；(2).酶切完成后，向離心管中加入等體積的pH為8.0的重蒸飽和酚，蓋緊管蓋，反復顛倒或用DNA混合儀轉動混勻，直至兩相混合均勻；(3).把離心管置于13000rpm下離心5分鐘，輕輕移動試管，用移液器將上清液體移至另一個尖底離心管中；(4).重復歩驟(2)(3);(5).向上述離心管中加入酚，再加入與酚等體積的氯仿或直接加入事先混好的1:1的酚-氯仿混合輛，蓋緊管蓋，反復顛倒或用DNA混合儀轉動混勻，至兩相混合均勻；(6).用移液器將上清液移至另一個尖底試管中；(7).向上述溶液中加入氯仿，蓋緊管蓋，反復顛倒或用DNA混合儀轉動混勻，至兩相混合均勻；(8).將上清液轉移至尖底試管中，加入一20。C的無水乙醇，顛倒混勻；(9).把離心管置入高速離心機，以13000rpm離心8分鐘，打開試管蓋,將試管口朝下，輕輕地倒掉上清液；(10).加入70%乙醇，清洗一次，把離心管放入高速離心機以13000卬m離心8分鐘，打開試管蓋,反轉試管口朝下,輕輕倒掉上清液；(11).將尖底管倒扣于吸水紙上，吸干殘留液體后平臥，室溫晾干；(12).晾干后加入雙蒸水，37'C保溫或常溫下過夜，使DNA溶解；(13).將溶解后的DNA進行濃度測定，然后置于一20'C冰凍保存。所述含有240ug/ml蛋白酶K的緩沖液為含有20mraol/LTris.Cl(pH8.0)、200mmol/LEDTA(pH8.0)、19dSDS、240ug/ml蛋白酶K的水溶液。本發明的提取方法省略了可致白細胞丟失的裂解紅細胞過程，利用特制DNA提取溶液直接對全血成份進行消化處理，然后對消化液用酚、酚-氯仿混合液以及氯仿進行抽提，減少了白細胞的損失，大大提高了DNA的產量，基本做到無損失全量提取DNA。在有些情況下，DNA是一種極為寶貴的、不可再生的資源，尤其是那些已經去世的具有寶貴研究價值的遺傳病患者，所留下的血液樣本量極為有限，若采用常規的DNA的提取方法提取DNA，由于提取率低，會造成不可估量的損失，而采用本發明的方法則可取得較好的效果。本發明具有效果好、操作簡單等優點。具體實施方式實施例提取全血DNA，步驟如下(1).取凍融處理的ACD(檸檬酸、檸檬鈉、葡萄糖)抗凝血100ul，置于1.5M離心試管中，加入400ul含有240ug/ml蛋白酶K的緩沖液，振蕩混勻，置于50'C水浴24小時，進行酶切，水浴過程中緩慢搖動離心管。(2).i切完成后，含有血液的混濁的溶液會變成清澈透明的紅葡萄酒狀，這時向離心管中加入等體積(約500ul)的PH為8.0重蒸飽和酚，蓋緊管蓋，反復顛倒20次左右或用DNA混合儀轉動混勻10分鐘，直至兩相混合均勻，切忌使用振蕩器或用力過猛。(3).把離心管置于13000rpm下離心5分鐘，輕輕移動試管，避免攪動界面，用lml吸頭的移液器將上清液體移至另一個1.5ml的尖底離心管中，為了最大限度回收DNA，可以稍稍吸帶下層的酚。(4).重復歩驟(2)(3)。(5).向上述離心管中加入250ul的酚，再加入250ul氯仿或直接加入500ul事先混好的1:1的酚-氯仿混合物，蓋緊管蓋，反復顛倒20次左右或用DNA混合儀轉動混勻10分鐘，直至兩相混合均勻。(6).用lml吸頭的移液器將上清液移至另一個1.5ml的尖底試管中，不要吸入下層的酚。(7).向上述溶液中加入500ul氯仿，蓋緊管蓋，反復顛倒20次左右或用DNA混合儀轉動混勻10分鐘，直至兩相混合均勻。(8).將上清液轉移至L5ml的尖底試管中，加入lml—20"C的無水乙醇，顛倒混勻，這時會看到呈棉絮狀析出的DNA。(9).把離心管置入高速離心機，以13000rpm離心8分鐘，打開試管蓋，將試管口朝下，輕輕地倒掉上清液。(10).加入lml70^乙醇，清洗一次，把離心管放入高速離心機以13000rpm離心8分鐘，打開試管蓋,反轉試管口朝下，輕輕倒掉上清液。(11).將尖底管倒扣于吸水紙上，吸干殘留液體后平臥，室溫晾干10—15分鐘。(12).晾干后加入雙蒸水(ddH20)100—500ul，37'C保溫3小時或常溫下過夜，使DNA溶解。(13).將溶解后的DNA進行濃度測定，然后置于一2(TC冰凍保存。所述含有240ug/ml蛋白酶K的緩沖液為含有20mmol/LTris.Gl(pH8.0)、200mmol/LEDTA(pH8.0)、1%SDS、240ug/ml蛋白酶K的水溶液。采用傳統酚氯仿提取法提取全血DNA，與本發明方法提取相同血樣DNA，lml血得到DNA的量的比較應用獨立樣本T檢驗，兩總體樣本均為II型糖尿病病人血樣，統計結果如下-血樣種類樣本量lml血得到DNA量的均數(Ug)標準差標準誤傳統酚、氯仿法29334.1583231.121731.81815本發明方法96246.36400123.4660112.60120設置置信水平為0=0.05,臨界置信水平為0.000,遠小于設置的置信水平，因此兩種方法提取DNA平均lml血得到DNA的量有顯著性差異。由數據可知，用本發明的提取方法的提取率高，幾乎Bj達l(Xm。由表中數據可知，本發明的方法所提取的DNA與傳統方法在0D260/280的比值方面略有下降，然而這并不影響所獲得的DNA分子在常規的分子生物學操作的實驗結果，因此這種變化是可以忽略的。所用樣本數據見下表l、2。表1.傳統酚、氯<table>tableseeoriginaldocumentpage6</column></row><table>取糖尿病患者血樣DNA試驗用血量lml全血提取DNA<table>tableseeoriginaldocumentpage6</column></row><table>175166.19216.61920.533.23841.17617667.0886.70880.513.41761.062177106.82410.68240.521.36481.32617877.9367.79360.515.58721.13818081.928.1920.516.3841.09119689.0328.90320.517.80641.296197.112.6%11.26960.522.53921.14419912512.50.5251.159201121.65612.16560.524.33121.032205106.96810.69680.521,39361.302215141.79214.17920.528.35840.95121876.67.660.515.321.21922191.1689.11680.518.23361.09822879.8247.98240.515.96481.282232106.65610.66560.521.33121.34524968.1366.81360.513.62721.085250216.46421.64640.543.29281.559252112.87211.28720.522.57441.161253163.01616.30160.532.60321.33125571.1847.11840.514.23681.36125756.7685.67680.511.35361.045263199.39219.93920.539.87841.46926776.2727.62720.515.25441.22427171.5287.15280.514.30561.34128550.0245.00240.510.00481.12628857.3685.73680.511.47361.242292187.26418.72640.537.45281.62229464.7766.47760.512.95521.54630665.2326.52320.513.04641.380311247.25624.72560.549.45121.493312107.81610.78160.5Si-56321.17731958.6485.86480.5ll.72961.35532252.45.240.510.481.39332364.7286.47280.512.94561.221330127.7212.7720.525.5441.073348241.07224.10720.548.21440.921350287.4828.7480.5574960.922354118.31211.83120.523.66241.5635764.3686.43680.512.8736L033384346.78434.67840.569.35681.65440265.6326.56320.513.12641.11240365.6086.56080.513.12161.89640890.2889.02880.518.05761.45143582.3048.23040.516.46081,374442122.34412.23440.524.46880.91744684.8568.48560.516.97121.27944848.9924.89920.59.79841.00745956.7765.67760.511.35521.237471102.68810.26880.520.53761.161478110.03211.00320.522.00641.173489102.28810.22880.520.45761.25249750.1445.01440.510.02881.18550571.4167.14160.514.28321.0750753.1685.31680.510,63361.09511118.21611.82160.523.64321.32651877.2647.72640.515.45281.115519117.60811.76080.523.52161.4524357.71235.77120.571.54241.74352861.726.1720.512.3441.907529105.36810.53680.521.07361.287531144.67214.46720.528.93441.11953358.1845.81840.511.63681.13653469.3526.93520.513.87041.09254164.086.4080.512.8161.116564176.95217.69520.535.39041.479575120.48812.04880.524.09761.503580231.78423.17840.546.35681.092592405.86440.58640.581.17281.116609193.519.350.538.71.66616129,512.950.525.91.46621203.520.350.540.71.47622737.30.514.61.54628828.20.516.41.21629343.534.350.568.71.52634216.521.650.543.31.32635818.10.516.21.33655174.517.450.534.91.6661656.50.5131.4267870.8967.08960.514.17921.088688180180.5361.3969315415.40.530.81.26700462.446.240.592.481.64775174.57.450.514.91.5275972.57.250.514.51.4176292.59.250.518.51.6576350.55.050.510.11.6076455.55.550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.36844.336816396.94439.694420171.1617.11648154.21615.421667154.71215.471282152.31215.231285133.82413.382493184.38418.438495163.50416.350499187.67218.7672101206.7220.672105182.51218.2512106"208.8420.884165289.58428.9584167157.82415.7824183202.07220.2072186245.01624.501620598.9289.8928211258.07225.8072225477.55247.7552234212.70421.2704235237.94423.794423758.765.876238212.34421.2344239114.37611.4376240145.70414.5704糖尿病患者血樣DNA本試驗用血lml全血提取260/280量(ml)DNA的量(ug)0.1427.5921.0150.1443.3681.0290.1396.9441.0250.1171.161.0040.1154.2161.0450.1154.7121.0220.1152.3121.0220.1133,8241.0370.1184.3841.0450.1163,5041.0430.1187.6721.0610.1206.721.2150.1182.5121.0760.1208.841.0810.1289.5841.0540.1157.8241.0560.1202.0721.0330.1245.0161.0450.198.9281.0790.1258.0721.5560.1477.5521.0620.1212.7041.2170.1237.9441.0690.158.761.1720.1212.3441.2860.1114.3761.0450.1145.7041.04224817917.90.11791.078277307.47230.74720.1307.4721.024281111.10411.11040.1111.1041.108282234.66423.46640.1234.6641.032283271.5627.1560.1271.561.027284181.68818.16880.1181.6881.196290430.30443.03040.1430.3041.433291212.87221.28720.1212,8721.038296249.14424.91440.1249.1441.086300466.77646.67760.1466.7761.487301405.68840.56880.1405.6881.244302255.75225.57520.1255.7521.026303334.21633.42160.1334.2161.034304149.08814.90880.1149.0881.086305110.59211.05920.1110.5921.061307200.35220.03520.1200.3521.025308162.6416.2640.1162.641.034309178.92817.89280.1178.9281.084316500.00850.00080.1500.0081.108318182.49618.24960.1182.4961.095320200.5620.0560.1200.561.025326205.43220.54320.1205.4321.047328116.52811.65280.1116.5281.019329178.417.840.1178.41.103331303.9230.3920.1303.921,042344292.85629.28560.1292.8560.994345298.60829.86080.1298.6081.15136585.768.5760.185.761.29367151.23215.12320.1151.2321.039369147.8414.7840.1147.841.077370190.13619.01360.1190.1361.032371197.35219.73520.1197.3521.025372338.71233.87120.1338.7121.064373420.34442.03440.1420.3441.058376655.40865.54080.1655.4081.047377330.8833.0880.1330.881.032378266.55226.65520.1266.5521.025379280.03228.00320.1280.0321.04138077.5047.75040.177.5041.229382127.82412.78240.1127.8241.209383499.49649.94960.1499.4961.109385513.251.320.1513.21.114386486.53648.65360.1486.5361.069389450.5645.0560.1450.561.186390451.99245.19920.1451.9921.174391157.1615.7160.1157.161.053394175.12817.51280.1175.1281.045395162.20816.22080.1162.2081.112396、273.6427.3640.1273.641.364398125.54412.55440.1125.5441.128399155.77615.57760.1155.7761.031404215.84821.58480.1215.8481.087421201.99220.19920.1201.9921.11442280.1768.01760.180.1761.102423242.824.280.1242.81.178424232.0423.2040.1232.040.978428173.8817.3880.1173.881.075498599.70459.97040.1599.7041.047500321.37632.13760.1321.3761.008501200.6820.0680.1200.681.005522243.0424.3040.1243.041.095525214.26421.42640.1214.2641.076527214.2421.4240.1214.241.325530183.16818.31680.1183.1681.053532104.1610.4160.1104.160.992535370.9237.0920.1370.921.01536179.26417.92640.1179.2640.986539276.26427.62640.1276.2641.007545277.02427.70240.1277.0240.97權利要求1.一種全血DNA的提取方法，其特征在于包括以下步驟(1).取凍融處理的ACD抗凝血，置于離心試管中，加入含有240ug/ml蛋白酶K的緩沖液，振蕩混勻，置于50℃水浴20～28小時，水浴過程中緩慢搖動離心管；(2).酶切完成后，向離心管中加入等體積的pH為8.0的重蒸飽和酚，蓋緊管蓋，反復顛倒或用DNA混合儀轉動混勻，直至兩相混合均勻；(3).把離心管置于13000rpm下離心5分鐘，輕輕移動試管，用移液器將上清液體移至另一個尖底離心管中；(4).重復步驟(2)(3)；(5).向上述離心管中加入酚，再加入與酚等體積的氯仿或直接加入事先混好的1∶1的酚-氯仿混合物，蓋緊管蓋，反復顛倒或用DNA混合儀轉動混勻，至兩相混合均勻；(6).用移液器將上清液移至另一個尖底試管中；(7).向上述溶液中加入氯仿，蓋緊管蓋，反復顛倒或用DNA混合儀轉動混勻，至兩相混合均勻；(8).將上清液轉移至尖底試管中，加入-20℃的無水乙醇，顛倒混勻；(9).把離心管置入高速離心機，以13000rpm離心8分鐘，打開試管蓋，將試管口朝下，輕輕地倒掉上清液；(10).加入70％乙醇，清洗一次，把離心管放入高速離心機以13000rpm離心8分鐘，打開試管蓋，反轉試管口朝下，輕輕倒掉上清液；(11).將尖底管倒扣于吸水紙上，吸干殘留液體后平臥，室溫晾干；(12).晾干后加入雙蒸水，37℃保溫或常溫下過夜，使DNA溶解；(13).將溶解后的DNA進行濃度測定，然后置于-20℃冰凍保存。2.根據權利要求1所述的一種全血DNA的提取方法，其特征在于:所述緩沖液為含有20ramol/LTris.Cl、200mmol/LEDTA、1%SDS、240ug/ml蛋白酶K的水溶液。全文摘要本發明公開了一種全血DNA的提取方法，解決了目前提取全血DNA時提取率不高的缺點，本發明的步驟為將血置于離心管中，加入含有蛋白酶K的緩沖液進行酶切，離心，加入酚，再離心，然后用酚-氯仿混合物提取2～3次，即得DNA；本發明所用的蛋白酶K的緩沖液為含有20mmol/LTris.Cl(pH8.0)、200mmol/LEDTA(pH8.0)、1％SDS、240ug/ml蛋白酶K的水溶液。本發明具有簡單、有效、提取率高等優點。文檔編號C12N15/10GK101215558SQ20081001377公開日2008年7月9日申請日期2008年1月15日優先權日2008年1月15日發明者劉文敏,鵬周,朱海寧,楊樹林,林汝湘,萌欒,溫曉燕,博王,剛陳,星陳,高春義,然魏申請人:剛陳