广东11选5

PCR技术原理、实验步骤和应用

一、实验目的

1.ZHANGWOJUHEMEILIANSHIFANYINGDEYUANLI。

2. 掌握移液枪和PCR仪的基本操作技术。

二、实验原理

PCRJISHU,JIJUHEMEILIANFANYING(polymerase chain reaction,PCR)SHIYOUMEIGUOPE CetusGONGSIDEKary MullisZAI1983NIAN(1993NIANHUONUOBEIERHUAXUEJIANG)JIANLIDE。ZHEIXIANGJISHUKEZAISHIGUANNEIDEJINGSHUXIAOSHIFANYINGJIUJIANGTEDINGDEDNAPIANDUANKUOZENGSHUBAIWANBEI,ZHEIZHONGXUNSUHUOQUDALIANGDANYIHESUANPIANDUANDEJISHUZAIFENZISHENGWUXUEYANJIUZHONGJUYOUJUZUQINGZHONGDEYIYI,JIDADITUIDONGLESHENGMINGKEXUEDEYANJIUJINZHAN。TABUJINSHIDNAFENXI**CHANGYONGDEJISHU,ERQIEZAIDNAZHONGZUYUBIAODA、JIYINJIEGOUFENXIHEGONGNENGJIANCEZHONGJUYOUZHONGYAODEYINGYONGJIAZHI。

PCRKEYIBEIRENWEISHIYUFASHENGZAIXIBAONEIDEDNAFUZHIGUOCHENGXIANGSIDEJISHU,QIJIEGUODOUSHIYIYUANLAIDEDNAWEIMOBANCHANSHENGXINDEHUBUDNAPIANDUAN。XIBAOZHONGDNADEFUZHISHIYIGEFEICHANGFUZADEGUOCHENG。CANYUFUZHIDEYOUDUOZHONGYINSU。PCRSHIZAISHIGUANZHONGJINXINGDEDNAFUZHIFANYING,JIBENYUANLIYUXIBAONEIDNAFUZHIXIANGSI,DANFANYINGTIXIXIANGDUIJIAOJIANDAN。

广东11选5 PCRYOUBIANXING--TUIHUO--YANSHENSANGEJIBENFANYINGBUZHOUGOUCHENG:①MOBANDNADEBIANXING:MOBANDNAJINGJIARE**94℃ZUOYOUYIDINGSHIJIANHOU,SHIMOBANDNASHUANGLIANHUOJINGPCRKUOZENGXINGCHENGDESHUANGLIANDNAJIELI,SHIZHICHENGWEIDANLIAN,YIBIANTAYUYINWUJIEHE,WEIXIALUNFANYINGZUOZHUNBEI;

②MOBANDNAYUYINWUDETUIHUO(FUXING):MOBANDNAJINGJIAREBIANXINGCHENGDANLIANHOU,WENDUJIANG**55℃ZUOYOU,YINWUYUMOBANDNADANLIANDEHUBUXULIEPEIDUIJIEHE;

③YINWUDEYANSHEN:DNAMOBAN--YINWUJIEHEWUZAITaqMEIDEZUOYONGXIA,YIdNTPWEIFANYINGYUANLIAO,BAXULIEWEIMOBAN,ANJIANJIPEIDUIYUBANBAOLIUFUZHIYUANLI,HECHENGYITIAOXINDEYUMOBANDNA LIANHUBUDEBANBAOLIUFUZHILIAN。

ZHONGFUXUNHUANBIANXING--TUIHUO--YANSHENSANGUOCHENG,JIUKEHUODEGENGDUODE“BANBAOLIUFUZHILIAN”,ERQIEZHEIZHONGXINLIANYOUKECHENGWEIXIACIXUNHUANDEMOBAN。MEIWANCHENGYIGEXUNHUANXU2~4FENZHONG, 2~3XIAOSHIJIUNENGJIANGDAIKUOMUDEJIYINKUOZENGFANGDAJIBAIWANBEI。

三、实验试剂与器材

MOBANDNA、2.5mmol/L dNTP

Taq DNAJUHEMEI(5U/μL)、SSRYINWU

10 ×buffer、15mmol/L Mg2+、ddH2O

广东11选5 PCRYI、YIYEQIANG、PCRBAN

四、实验步骤

1、配制20μL反应体系,在PCR板中依次加入下列溶液:

广东11选5 MOBANDNA    2μL

YINWU1      1μL

YINWU2      1μL

广东11选5 dNTP      1.5μL

广东11选5 MgCl2        2μL

广东11选5 10×buffer    2μL

广东11选5 ddH2O      10μL

广东11选5 TaqMEI      0.5μL

2、设置PCR反应程序。

3、上样,启动反应程序。

4、扩增产物的电泳检测。

附PCR技术应用

广东11选5 1、SHENGMINGKEXUE a、RENLEIJIYINZUJIHUA SUIZHEDEPCRRIZHENWANSHAN,KEXUEJIAYU2003NIANZAIWANCHENGDERENLEIJIYINZU“GONGZUOKUANGJIATU”DEJICHUSHANG,JINGGUOZHENGLI、FENLEIHEPAILIEHOUDEDAODEGENGJIAZHUNQUE、QINGXI、WANZHENGDEJIYINZUTUPU。ZHEISHIDUIRENLEIJIYINZUJIBENMIANMAODE**JIESHI,BIAOMINGKEXUEJIAMENKAISHIBUFEN“DU”CHURENLEISHENGMING“TIANSHU”SUOYUNHANDENEIRONG。 b、HOUJIYINZUJIHUA RENLEIJIYINZUDNAXULIETUPUWANCHENGHOU,JIANDINGJIYINZUDUOTAIXINGJIQIDANBEIXINGYIJIXUNZHAOQIZAISHENGWUHEYIXUEYINGYONGZHONGZHONGYAOCHENGWEIRENMENGUANXINDEREDIAN。YIYANJIUJIYINGONGNENGWEIHEXINDE“HOUJIYINZUSHIDAI”YIJINGLAILIN,DAGUIMODEJIEGOUJIYINZU、DANBAIZHIZUYIJIYAOWUJIYINZUDEYANJIUJIHUAYIJINGCHENGWEIXINDEREDIAN。 c、WUZHONGDEFENLEI、JINHUAJIQINYUANGUANXI KEYIJINXINGWUZHONGJINHUADEBAOSHOUXINGFENXIJIWUZHONGDUOTAIXINGFENXI、WUZHONGJIANDING。

2、YIYAO a、JIBINGDEZHENDUANHEZHILIAO YICHUANXINGJIBING:RUDIZHONGHAIPINXUE、LIANDAOZHUANGXIBAOPINXUE、NINGXUEYINZIQUEFADENGYOUYICHUANQINGXIANGDEJIBING,YOUQISHILAONIANXINGJIBING,RUTANGNIAOBING、GAOXUEZHIZHENG、SHEN**ZHONGLIUZHONGDEYIBUFEN,JUNKEYUCE;AIJIYINDEJIANCEHEZHENDUAN:JIANCEEXINGZHONGLIUDEBIAOJIWULAIZHENDUANAIZHENGDENGJIBING;LIYONGJIYINZHILIAOFANGFAZHILIAOZHONGLIUXINGJIBING。 b、ZHIBINGBINGYUANTIDEJIANCE JIANCEFANWEIBAOKUOXIJUN、BINGDU(SARSJIQINLIUGANBINGDUH5N1DENG)、YUANCHONGJIJISHENGCHONG、MEIJUN、LIKECITI、YIYUANTIHEZHIYUANTIDENGYIQIEWEISHENGWU。JIANCEDELINGMINDUHETEYIXINGDOUYUANGAOYUDANGQIANDEMIANYIXUEFANGFA,SUOXUSHIJIANYEYIDADAOLINCHUANGYAOQIU,ZHEIDUIYUNANYUPEIYANGDEBINGDU(YIGAN),XIJUN(RUJIEHE、YANYANGJUN)HEYUANCHONG(RUMEIDULUOXUANTI)DENGLAISHUOYOUWEISHIYONG。 c、DNAZHIWEN、GETISHIBIE(DNASHENFENZHENG)、QINZIGUANXIJIANBIEHEFAYIWUZHENG KEYIYONGYIGENTOUFA、YIGEXIBAO、YIGEJINGZILAIWANCHENGSHANGSHUGONGZUO,ZHEIYILINGYUYEYIFAZHANDAOGUSUIHUOZANGQIYIZHIPEIXING。 d、SHENGWUGONGCHENGZHIYAO XUDUOYAOWUKETONGGUOGONGCHENGJUNHEXIBAOLAIDALIANGSHENGCHAN,RUGANRAOSU、BAIJIESU、CUHONGXIBAOSHENGZHANGSUDENGYAOWU。 e、ZHUANJIYINDONGWUZHIYAOJIJIBINGMOXING ZHUANJIYINDONGWUYUYIXUEJISHENGWUYIYAOYANJIUDEGUANXIYUELAIYUEMIQIE。JINNIANLAI,GEZHONGRENLEIJIBINGZHUANJIYINDONGWUMOXINGBUDUANJIANLI。RUZHUANJIYINDONGWUZAIYICHUANBING、XINXUEGUANJIBING、ZHONGLIU、GAOXUEYABING、BINGDUXINGJIBING、YIZHONGYIZHI、SHUXUEYIXUE、YAOLIXUEYANJIUZHONGDEYINGYONG;LIYONGZHUANJIYINDONGWU-RUXIANSHENGWUFANYINGQISHENGCHANYAOWUDANBAI,HAOBIZAIDONGWUSHENSHANGJIAN“YAOCHANG”,KEYICONGDONGWURUZHIZHONGYUANYUANBUDUANDIHUODEJUYOUWENDINGSHENGWUHUOXINGDEJIYINCHANPIN。ZHEISHIYIZHONGQUANXINDEYAOWUSHENGCHANMOSHI,JUYOUTOUZICHENGBENDI,YAOWUYANZHIZHOUQIDUANHEJINGJIXIAOYIGAODENGYOUDIAN。

3、NONGYEKEXUE a、ZHUANJIYINZHIWU ANQIGONGNENGZHUYAOFENTIGAOCHANLIANG、KANGBINGLI、KANGCHUCAOJI、GAILIANGPINZHIHEFAYUDIAOJIE。RU:DADOU、YUMI、MALINGSHU、FANQIEDENG。 b、ZHUANJIYINDONGWU ZAINONGYEFANGMIAN,ZHUYAOZAIGAILIANGCHUQINSHENGCHANXINGZHUANG,TIGAOCHUQINKANGBINGLIJILIYONGZHUANJIYINCHUQINSHENGCHANFEICHANGGUICHUCHANPIN。

4、KAOGUXUEJILISHISHIJIANJIEDULIYONGRENLEIDUANCHUANLIANZHONGFUSTR-PCRJISHU,YANJIURENLEIZHONGZUDEYICHUANDUOTAIXING,XIAOGUOFEICHANGWENDING。MUQIANCIJISHUYIGUANGFANYONGYUSHENGWUKAOGU、ZHONGXIFAYU、MINZUXUE、RENLEIXUEHEKAOGUXUEDENGGEGELINGYUZHONG。

5、WEISHENGANQUAN a、SHIPINWEISHENGWUDEJIANCE CHUANTONGDEZHIBINGJUNJIANCESHOUXIANJINGGUOZHANGSHIJIANDEPEIYANG,FEISHIFEILI,YINGYONGPCRJISHUZEFEICHANGXUNSU、ZHUNQUE。ZHUYAOYONGYU:SHIPINZHIBINGJUNDEJIANCE,RUROUDUSUOJUNDENG;RUSUANJUNDEJIANCE;SHUIZHONGXIJUNZHIBIAOCEDING。 b、ZHUANJIYINSHIPINDEJIANCE MUQIANSHIJIEZHUANJIYINSHIPINYIJINGYOU100DUOWUZHONG,DADUOSHUYIJINGYONGYUSHIPIN。ZHUANJIYINSHIPINDUIRENTIJIANKANGDEWEIHAIJIDUISHENGTAIDEYINGXIANGYERISHOUSHIJIEGUANGFANDEZHONGSHI,YINCIDUIZHUANJIYINSHIPINDEJIANCECHENGWEIKONGZHIQIFANLANDEYIZHONGSHOUDUAN。 c、DONG、ZHIWUJIANYI LINGMIN、TEYI、KUAISUZHENDUANJIANCEFANGFASHIWOGUOJINCHUKOUKOUANDEMENWEI,JIANCHACHURU**DERENYUAN、DONG、ZHIWU(ZHONGCHU、ZHONGZI)DENGSHIFOUXIEDAILIEXINGCHUANRANBING(AIZI、DONGWUBINGDU、ZHIWUBINGDUDENG)BINGYUANTI,SHIPIN、SILIAODENGSHIFOUDAISHAMENSHIJUNDENGJUNXUYAOJIYINZHENDUANSHOUDUANJIANGZHEIXIEBINGJUNJUZHIYU**ZHIWAI,SHITIGAOWOGUOZONGHEGUOLIDEBIYAOBAOZHENG。